Chromatin Program & Abstract BookStructural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase ... Epigenetic aspects of lineage commitment

Structure&FunctionPunta Cana, Dominican Republic5 - 8 December 2006

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Organized By:Tony Kouzarides and Abcam

ChromatinProgram & Abstract Book

Punta Cana Prog 30/10/06 10:08 Page 2

Program & Abstract Book

The third

ChromatinStructure & Function

Punta Cana, Dominican Republic

5 - 8 December 2006

Organizers:Tony Kouzarides

(University of Cambridge)and Abcam

Table of contents

Conference Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 2

Poster Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 6

Abstracts - Oral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 18

Abstracts - Poster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 51

Resort Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 179

Disclaimer: Material contained within this booklet should be citied only with permission from the author(s).No live recording or photography is permitted during the oral or poster sessions.

Copyright © 2006 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.All information / detail is correct at time of going to print.

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Tuesday 5th DecemberMeeting room - Allegro Plaza

Keynote Speaker - introduced by Tony Kouzarides18:00 - 19:00 Steve Henikoff . . . . . . . . . . . . . . . . . . . . . . . .Page 18

Epigenetic patterns generated by histone replacement

Welcome reception and buffet at poolside.

Allegro Live resort show

Wednesday 6th DecemberChair: Jerry Workman

09:00 - 09:30 Yang Shi . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 19The identification of histone demethylases established thedynamic and reversible nature of histone methylationregulation

09:30 - 09:45 Jesper Christensen . . . . . . . . . . . . . . . . . . . . .Page 20The retinoblastoma tumor suppressor binding protein RBP2 isa transcriptional repressor demethylating tri- and dimethylatedlysine 4 on Histone H3

09:45 - 10:00 Mischa Machius . . . . . . . . . . . . . . . . . . . . . . .Page 21Structural basis for CoREST-dependent demethylation ofnucleosomes by the human LSD1 histone demethylase

10:00 - 10:30 Ramin Shiekhattar . . . . . . . . . . . . . . . . . . . . .Page 22Functional and biochemical characterization of histonedemethylase complexes

Drinks break in hotel lobby

11:00 - 11:30 Yi Zhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 23Histone demethylation by the JmjC domain-containing proteins

11:30 - 11:45 Francis Stewart . . . . . . . . . . . . . . . . . . . . . . . .Page 24Epigenetic aspects of lineage commitment

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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006

Conference Program


11:45 - 12:00 Henk Stunnenberg . . . . . . . . . . . . . . . . . . . . .Page 25Title and abstract unavailable

12:00 - 12:30 Ali Shilatifard . . . . . . . . . . . . . . . . . . . . . . . . . .Page 26H2B monoubiquitination and H3K4 methylation via COMPASS

Lunch at the Beach Buffet Restaurant and free time

Chair: Yang Shi

16:00 - 16:30 Paulo Sassone-Corsi . . . . . . . . . . . . . . . . . . .Page 27A chromatin remodeling clock

16:30 - 16:45 Sung Hee Baek . . . . . . . . . . . . . . . . . . . . . . .Page 28A Novel Link between SUMO Modification of a ChromatinRemodeling Complex and Cancer Metastasis

16:45 - 17:00 Laszlo Tora . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 29The simultaneously dimethylated Lys-9 and phosphorylatedSer-10 tails of histone H3 adopt different conformationsduring mitosis

17:00 - 17:30 Tony Kouzarides . . . . . . . . . . . . . . . . . . . . . . .Page 30Characterisation of novel histone modifications

18:00 - 21:00 Posters and buffet by the pool

Allegro Live resort show

Thursday 7th DecemberChair: Genevieve Almouzni

09:00 - 09:30 Thomas Jenuwein . . . . . . . . . . . . . . . . . . . . . .Page 31Epigenetic control by histone methylation

09:30 - 09:45 Roberta Benetti . . . . . . . . . . . . . . . . . . . . . . .Page 32The role of Dicer in the regulation of chromatin at telomeres

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Conference Program


09:45 - 10:00 Mareike Puschendorf . . . . . . . . . . . . . . . . . . .Page 33Ezh2 independent targeting of PRC1 proteins to paternalconstitutive heterochromatin in mouse pre-implantation embryos

10:00 - 10:30 Edith Heard . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 34The nuclear and epigenetic dynamics of X-chromosomeinactivation in the mouse


11:00 - 11:30 Adrian Bird . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 35MeCP2: molecular interactions and phenotypic stability in amouse model of Rett Syndrome

11:30 - 11:45 Jon Penterman . . . . . . . . . . . . . . . . . . . . . . . .Page 36DNA demethylation in Arabidopsis thaliana

11:45 - 12:00 Francois Fuks . . . . . . . . . . . . . . . . . . . . . . . . .Page 37The Polycomb Group protein EZH2 is recruited to promoters byMECP2

12:00 - 12:30 Shelley Berger . . . . . . . . . . . . . . . . . . . . . . . .Page 38Factor and histone covalent modifications in genome regulation

Lunch at the Beach Buffet Restaurant and free time

Chair: Ramin Shiekhattar

16:00 - 16:30 Danny Reinberg . . . . . . . . . . . . . . . . . . . . . . .Page 39A molecular understanding of epigenetics

16:30 - 16:45 Jessica Tyler . . . . . . . . . . . . . . . . . . . . . . . . . .Page 40The mechanistic basis for the requirement of promoterchromatin disassembly for transcriptional activation

16:45 - 17:00 Gratien Prefontaine . . . . . . . . . . . . . . . . . . . . .Page 41Epigenetic mechanisms influencing pituitary gene expression

17:00 - 17:30 Bob Kingston . . . . . . . . . . . . . . . . . . . . . . . . .Page 42Possible roles in silencing for piRNAs

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18:00 - 19:30 Posters and drinks by the pool

Beach barbeque and live band on the sand

Friday 8th DecemberChair: Edith Heard

09:00 - 09:30 Michael Grunstein . . . . . . . . . . . . . . . . . . . . . .Page 43Deacetylation of histone H4 K16 regulates gene activity in yeast

09:30 - 09:45 Ann Ehrenhofer-Murray . . . . . . . . . . . . . . . . .Page 44A role for the HDAC Rpd3 in establishing eurchromatin-heterochromatin boundaries at yeast telomeres

09:45 - 10:00 Wyatt Yue . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 45CARM1 and histone methylation - a structural study

10:00 - 10:30 Sharon Dent . . . . . . . . . . . . . . . . . . . . . . . . . .Page 46Common and unique factors regulate Set1-mediatedmethylation of the Dam1 kinetochore protein and histone H3


11:00 - 11:30 Genevieve Almouzni . . . . . . . . . . . . . . . . . . . .Page 47Chromatin assembly factors, histone H3 variants and cell cycle

11:30 - 11:45 Dmitry Fyodorov . . . . . . . . . . . . . . . . . . . . . . .Page 48ATP-dependant deposition of Histone H3.3 by Drosophila CHD1in vivo

11:45 - 12:00 Roberto Mantovani . . . . . . . . . . . . . . . . . . . . .Page 49The histone fold trimer NF-Y is required to define positivehistone marks in CCAAT-promotors: a genome wide analysis

12:00 - 12:30 Jerry Workman . . . . . . . . . . . . . . . . . . . . . . . .Page 50Histone modification and chromatin remodeling in transcription

Lunch at the Beach Buffet Restaurant

Conference ends

5

Conference Program


Abstract P1 Karl Agger . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 51The role of the polycomb group protein RYBP in oncogeneinduced senescence

Abstract P2 Helena Ahlfors . . . . . . . . . . . . . . . . . . . . . . . .Page 52A novel player in T helper cell differentiation

Abstract P3 Barbara Alberter . . . . . . . . . . . . . . . . . . . . . . .Page 53Histone modification pattern of the T lymphotropic Herpesvirussaimiri genome in latency

Abstract P4 Marco Alvarez . . . . . . . . . . . . . . . . . . . . . . . . .Page 54Histone variant macroH2A is an epigenetic factor involved inthe modulation of ribosomal gene expression during seasonaladaptation of carp fish

Abstract P5 Terra Arnason . . . . . . . . . . . . . . . . . . . . . . . . .Page 55Rsp5 is required for nuclear shuttling of the Snf1 kinasecomplex in yeast

Abstract P6 Stuart Atkinson . . . . . . . . . . . . . . . . . . . . . . . .Page 56Epigenetic mechanisms of pluripotency and differentiation

Abstract P7 Joanne Attema . . . . . . . . . . . . . . . . . . . . . . . .Page 57Epigenetic features of hematopoietic stem cells using smallnumbers of highly purified primary cells

Abstract P8 Kristin Baetz . . . . . . . . . . . . . . . . . . . . . . . . . .Page 58NuA4 is a cellular “Hub”: an integrative map of physical andgenetic interactions mediated by the NuA4 histoneacetyltransferase

Abstract P9 Slobodan Barbaric . . . . . . . . . . . . . . . . . . . . .Page 59Chromatin remodeling activities at the yeast PHO84 promoter

Abstract P10 Vivian Bardwell . . . . . . . . . . . . . . . . . . . . . . . .Page 60Polycomb group and SCF ubiquitin ligases are found in a novelBCOR complex that is recruited to BCL6 targets

Abstract P11 Amrita Basu . . . . . . . . . . . . . . . . . . . . . . . . . .Page 61Computational prediction of histone and non-histone proteins

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Poster Index


Abstract P12 Mark Bedford . . . . . . . . . . . . . . . . . . . . . . . . .Page 62Screening for the methylated proteome

Abstract P13 Sukesh R. Bhaumik . . . . . . . . . . . . . . . . . . . .Page 63Regulation of transcriptional activation by mRNA cap-bindingcomplex in vivo

Abstract P14 Marjorie Brand . . . . . . . . . . . . . . . . . . . . . . . .Page 64The Ash2L/MLL2 methyltransferase complex is important for ß-globin transcription during erythroid differentiation

Abstract P15 Lauren Buro . . . . . . . . . . . . . . . . . . . . . . . . . .Page 65Histone methylation patterns at interferon-gamma induciblegene loci

Abstract P16 Jill Butler . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 66CXXC-finger protein 1 regulates Dnmt1 protein expression

Abstract P17 Jim Cakouros . . . . . . . . . . . . . . . . . . . . . . . . .Page 67Identification of a novel enzyme which regulates the kinetics ofhistone arginine methylation in Drosophila melanogaster

Abstract P18 Raymond Camahort . . . . . . . . . . . . . . . . . . . .Page 68Genome-wide analysis of the budding yeast histone variantCse4 reveals occupancy at a single centromeric nucleosome aswell as additional non-centromeric locations

Abstract P19 Dylan Carney . . . . . . . . . . . . . . . . . . . . . . . . .Page 69The RAG2 PHD Finger links the histone code to V(D)Jrecombination

Abstract P20 Beverly Chilton . . . . . . . . . . . . . . . . . . . . . . . .Page 70Analysis of RUSH/SMARCA3 isoforms and their interactionswith Egr-1 and c-Rel in the regulation of transcription

Abstract P21 Alexandra Chittka . . . . . . . . . . . . . . . . . . . . . .Page 71Signalling by a novel p75 neurotrophin receptor interactingprotein, SC1/PRDM4

Abstract P22 Leslie Chu . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 72Inheritance of epigenetic chromatin states

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Poster Index


Abstract P23 Mair Churchill . . . . . . . . . . . . . . . . . . . . . . . . .Page 73Structural basis for the histone chaperone activity of Asf1

Abstract P24 Jeffrey Craig . . . . . . . . . . . . . . . . . . . . . . . . . .Page 74What makes centromeres localise and cluster in interphasenuclei?

Abstract P25 Valerie Crusselle-Davis . . . . . . . . . . . . . . . . . .Page 75Regulation of beta-globin expression through the recruitmentof chromatin modifying enzymes by TFII-I and USF

Abstract P26 Eullia de Nadal . . . . . . . . . . . . . . . . . . . . . . . .Page 76Control of gene expression by the yeast Hog1 MAPK.

Abstract P27 Foteini Davrazou . . . . . . . . . . . . . . . . . . . . . . .Page 77Molecular mechanism of histone H3K4me3 recognition by thePHD finger of ING2

Abstract P28 Roger Deal . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 78Repression of flowering in Arabidopsis requires histone H2A.Zdeposition by a putative SWR1 complex

Abstract P29 Laurent Delva . . . . . . . . . . . . . . . . . . . . . . . . .Page 79The Transcription Intermediary Factor 2 is required forzebrafish development

Abstract P30 Luisa Di Stefano . . . . . . . . . . . . . . . . . . . . . .Page 80Lsd1 mutation in Drosophila disrupt normal level of H3K4methylation and affects viability and fertility

Abstract P31 Stephan Diekmann . . . . . . . . . . . . . . . . . . . . .Page 81In vivo dynamic (FRAP, FCS) and neighbourhood relation (AB-FRET, FLIM) studies of human inner kinetochore proteins

Abstract P32 Jeffrey Dilworth . . . . . . . . . . . . . . . . . . . . . . . .Page 82MEF2 helps establish muscle specific pattern of geneexpression by recruiting Trithorax Group proteins to specificpromoters

Abstract P33 Ivana Djuretic . . . . . . . . . . . . . . . . . . . . . . . . .Page 83T-bet and Runx3 cooperate to activate Interferon gamma andsilence Interleukin-4 in T helper-1 cells

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Abstract P34 Tom Donndelinger . . . . . . . . . . . . . . . . . . . . . .Page 84Seeing cells in a new light: Improving resolution with ascientific approach to tissue processing

Abstract P35 Bojan Drobic . . . . . . . . . . . . . . . . . . . . . . . . . .Page 85Characterization of Histone H3 kinases, MSK1 and MSK2

Abstract P36 Danielle Ellis . . . . . . . . . . . . . . . . . . . . . . . . . .Page 86Histone acetylation of SRC and p21 promoters in response tohistone deacetylase inhibitor treatment; implications of HDACactivity and SRC expression

Abstract P37 Alexander Erkine . . . . . . . . . . . . . . . . . . . . . .Page 87Differential mechanisms of nucleosome displacement at yeastheat shock gene promoters

Abstract P38 Ragnhild Eskeland . . . . . . . . . . . . . . . . . . . . .Page 88HP1 binding to chromatin methylated at H3K9 is enhanced byauxiliary factors

Abstract P39 George Feehery . . . . . . . . . . . . . . . . . . . . . . .Page 89CpG methylated DNA standards and control primers for use inmethyl sensitive PCR and bisulphite sequencing

Abstract P40 Barna Fodor . . . . . . . . . . . . . . . . . . . . . . . . . .Page 90Identification of novel pericentric proteins by their localization

Abstract P41 Maria Fousteri . . . . . . . . . . . . . . . . . . . . . . . . .Page 91Cockayne syndrome A and B proteins differentially regulaterecruitment of chromatin remodeling and repair factors tostalled RNA polymerase II in vivo

Abstract P42 Robert Gillespie . . . . . . . . . . . . . . . . . . . . . . .Page 92Retinoid regulated association of transcriptional coregulatorsand the polycomb group protein SUZ12 with the retinoic acidresponse elements of Hoxa1, RARß2, and Cyp26A1 in F9embryonal carcinoma cells

Abstract P43 Clara Goday . . . . . . . . . . . . . . . . . . . . . . . . . .Page 93Chromatin modifications in germline chromosomes of sciarid flies

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Poster Index


Abstract P44 Aaron Goldberg . . . . . . . . . . . . . . . . . . . . . . .Page 94HIRA-dependent incorporation of histone H3.3 marks activegenes in mouse embryonic stem cells

Abstract P45 Elizabeth Goneska . . . . . . . . . . . . . . . . . . . . .Page 95Phosphorylation of the SQ H2A.X motif is required for propermeiosis and mitosis in Tetrahymena thermophila

Abstract P46 Susana Gonzalo . . . . . . . . . . . . . . . . . . . . . . .Page 96Telomere epigenetic modifications: a control of telomere lengthand a stop on recombination

Abstract P47 Tanya Gustafson . . . . . . . . . . . . . . . . . . . . . . .Page 97Epigenetic silencing of Singleminded-2 in breast cancer

Abstract P48 Soon-Ki Han . . . . . . . . . . . . . . . . . . . . . . . . . .Page 98Role of plant CBP/p300-like genes in the regulation offlowering time

Abstract P49 Christin Hanigan . . . . . . . . . . . . . . . . . . . . . . .Page 99Identification of an HDAC2 mutation in colorectal cancer andits consequences

Abstract P50 Troy Harkness . . . . . . . . . . . . . . . . . . . . . . . .Page100Rsp5 is required for nuclear shuttling of the Snf1 kinasecomplex in yeast

Abstract P51 Tiffany Hung . . . . . . . . . . . . . . . . . . . . . . . . .Page 101ING4 recognition of histone H3 trimethylated at lysine 4

Abstract P52 David Johnson . . . . . . . . . . . . . . . . . . . . . . .Page 102E2F1 and GCN5 facilitate the recruitment of nucleotide excisionrepair factors to sites of UV-induced DNA damage

Abstract P53 Paul Kalitsis . . . . . . . . . . . . . . . . . . . . . . . . .Page 103Nucleosome spacing analysis of repeat DNA regions in themouse genome

Abstract P54 Min-Jeong Kang . . . . . . . . . . . . . . . . . . . . . .Page 104Role of a RPD3/HDA1 family histone deacetylase in the regulationof phytochrome-mediated light respases in Arabidopsis

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Poster Index

Abstract P55 Panagiota Karagianni . . . . . . . . . . . . . . . . . .Page 105ICBP90, a putative link between histone ubiquitination and cellcycle progression

Abstract P56 Emmanuel Kas . . . . . . . . . . . . . . . . . . . . . . .Page 106Altering the structure and functional properties ofheterochromatin with satellite-specific minor-groove binders

Abstract P57 Chul Geun Kim . . . . . . . . . . . . . . . . . . . . . . .Page 107PIAS1 confers erythroid cell specific αα-globin gene regulationby the CP2 transcription factor family

Abstract P58 Keun Il Kim . . . . . . . . . . . . . . . . . . . . . . . . . .Page 108A novel link between SUMO modification of a chromatinremodeling complex and cancer metastasis

Abstract P59 Sarah Kimmins . . . . . . . . . . . . . . . . . . . . . . .Page 109Methylation of Histone H3 at lysine 4 is dynamic and tightlyregulated during male germ cell development

Abstract P60 Robert Klose . . . . . . . . . . . . . . . . . . . . . . . . .Page 110JmjC-domain-containing proteins and histone demethylation

Abstract P61 Christoph Koch . . . . . . . . . . . . . . . . . . . . . . .Page 111The landscape of activating histone modifications across 1% ofthe human genome

Abstract P62 Ryoki Kujiki . . . . . . . . . . . . . . . . . . . . . . . . . .Page 1121alpha,25(OH)2D3-induced transrepression on 1alpha-hydroxylase gene promoter mediates chromatin remodelingthrough WINAC

Abstract P63 Sharmistha Kundu . . . . . . . . . . . . . . . . . . . .Page 113SWI/SNF establishes transcriptional memory at theSaccharomyces cerevisiae GAL1 gene

Abstract P64 Georg Kustatscher . . . . . . . . . . . . . . . . . . . .Page 114Metabolite-sensitive and metabolite-insensitive chromatinsurfaces through the human histone macroH2A

Abstract P65 Hyockman Kwon . . . . . . . . . . . . . . . . . . . . . .Page 115BAF53-dependent higher-order chromatin structure as thecompartment of replication and repair foci


Abstract P66 Monika Lachner . . . . . . . . . . . . . . . . . . . . . .Page 116Studying lysine methylation in non-histone proteins

Abstract P67 Brian Larsen . . . . . . . . . . . . . . . . . . . . . . . . .Page 117Caspase 3 mediated DNA strand breaks contribute to genomicreorganization during skeletal muscle terminal differentiation

Abstract P68 Richard Lawrence . . . . . . . . . . . . . . . . . . . . .Page 118Mechanisms controlling dynamic Swi6/HP1 binding in S.pombe facilitate de novo heterochromatin formation

Abstract P69 Frederic Leduc . . . . . . . . . . . . . . . . . . . . . . .Page 119Presence of gamma-H2AX in elongating spermatids:involvement of NHEJ?

Abstract P70 Min Gyu Lee . . . . . . . . . . . . . . . . . . . . . . . . .Page 120Functional association of a trimethyl H3K4 demethylase andRing6a/MBLR, a polycomb-like protein

Abstract P71 Niraj Lodhi . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 121Histone acetylation (H3K9) and methylation (H3K4) of thenucleosome over core promoter are associated with theinduction of tobacco PR-1a gene

Abstract P72 Mattias Mannervik . . . . . . . . . . . . . . . . . . . .Page 122An HDAC3/SMRTER/Ebi complex required for Snail repressorfunction in Drosophila development

Abstract P73 Robert Martin . . . . . . . . . . . . . . . . . . . . . . . .Page 123Chromatin labeling and distribution in living cells

Abstract P74 Peter McKeown . . . . . . . . . . . . . . . . . . . . . . .Page 124Chromatin components of the Arabidopsis thaliana nucleolus

Abstract P75 Rosalind Meldrum . . . . . . . . . . . . . . . . . . . .Page 125Visualisation of DNA repair and chromatin dynamics

Abstract P76 Brendon Monahan . . . . . . . . . . . . . . . . . . . .Page 126Purification and characterization of the fission yeast Swi/Snfand RSC chromatin remodeling complexes

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Abstract P77 Antonin Morillon . . . . . . . . . . . . . . . . . . . . . .Page 127Transcriptional co-suppression in S. cerevisiae

Abstract P78 Ashby Morrison . . . . . . . . . . . . . . . . . . . . . . .Page 128Mec1/Tel1-dependent phosphorylation of a chromatinremodeling complex influences the DNA damage checkpointpathway

Abstract P79 Raul Mostoslavsky . . . . . . . . . . . . . . . . . . . .Page 129Genomic instability and aging-like phenotype in the absence ofmammalian SIRT6

Abstract P80 Takahiro Nakayama . . . . . . . . . . . . . . . . . . .Page 130Drosophila GAGA factor promotes histone H3.3 replacementthat prevents the heterochromatin spreading

Abstract P81 Zuyao Ni . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 131The tumor suppressor BRG1 silences the distal silencers atinterferon-responsive genes

Abstract P82 Olivia Osborn . . . . . . . . . . . . . . . . . . . . . . . .Page 132Transcriptional targets of Af4

Abstract P83 Julia Pagan . . . . . . . . . . . . . . . . . . . . . . . . . .Page 133A novel corepressor, BCOR-L1, functions through CTBP andclass 2 HDACs

Abstract P84 Maria Panchenko . . . . . . . . . . . . . . . . . . . . .Page 134Role of Jade-1 in the HAT HBO1 complex

Abstract P85 Tej Pandita . . . . . . . . . . . . . . . . . . . . . . . . . .Page 135Mammalian ortholog of Drosophila MOF is critical forembryogenesis and DNA repair

Abstract P86 Maëlle Pannetier . . . . . . . . . . . . . . . . . . . . . .Page 136Imprinting perturbation in mouse hepatocarcinoma: linkbetween DNA methylation and histone methylation

Abstract P87 Janet Partridge . . . . . . . . . . . . . . . . . . . . . . .Page 137Establishment and maintenance of centromericheterochromatin in fission yeast are functionally separable

13

Poster Index


14


Abstract P88 Kelly Perkins . . . . . . . . . . . . . . . . . . . . . . . . .Page 138Activated HIV-1 provirus forms a gene loop, connecting viraltranscriptional initiation with termination

Abstract P89 David Picketts . . . . . . . . . . . . . . . . . . . . . . . .Page 139SNF2L-mediated control of cell number in the developing brain

Abstract P90 Romina Ponzielli . . . . . . . . . . . . . . . . . . . . . .Page 140Optimization of experimental design parameters of ChIP-on-chip studies

Abstract P91 Ryan Raisner . . . . . . . . . . . . . . . . . . . . . . . .Page 141Single nucleosome resolution mapping of the histone variantH2A.Z in a developing organism

Abstract P92 Rama Natarajan . . . . . . . . . . . . . . . . . . . . . .Page 142Genome-wide analysis of histone lysine methylation variationscaused by diabetic conditions in human monocytes

Abstract P93 Edward Ramos . . . . . . . . . . . . . . . . . . . . . . .Page 143Global characterization and function of Gypsy-like endogenousinsulators in Drosophila melanogaster

Abstract P94 William Renthal . . . . . . . . . . . . . . . . . . . . . . .Page 144Class II histone deacetylases regulate the behavioraladaptations to chronic cocaine and stress

Abstract P95 Karsten Rippe . . . . . . . . . . . . . . . . . . . . . . . .Page 145Activities of histone chaperone NAP1: Association states andinteractions with histones, nucleosome assembly and effect onthe chromatin fiber conformation

Abstract P96 Charles Roberts . . . . . . . . . . . . . . . . . . . . . .Page 146The Swi/Snf chromatin remodeling complex regulates lineagespecific transcription programs during development andimpairment of this activity causes cancer

Abstract P97 Paul Sadowski . . . . . . . . . . . . . . . . . . . . . . .Page 147Post-translational modification of the insulator protein, CTCF

Abstract P98 Teresa Sanchez Alcaraz . . . . . . . . . . . . . . . .Page 148Role of USP7 and GMP synthetase in deubiquitination ofhuman histone H2B


Abstract P99 Annette Scharf . . . . . . . . . . . . . . . . . . . . . . .Page 149Dynamics of histone modifications during chromatin assembly

Abstract P100 Stefan Schoeftner . . . . . . . . . . . . . . . . . . . . .Page 150Screening for miRNAs regulating mammalian telomeres

Abstract P101 Gunnar Schotta . . . . . . . . . . . . . . . . . . . . . .Page 151A genome-wide transition to H4K20 mono-methylation impairsstress-induced and programd DNA damage response in themouse

Abstract P102 David Schrump . . . . . . . . . . . . . . . . . . . . . . .Page 152Brother of the Regulator of Imprinted Sites (BORIS) recruitsSp1 to modulate NY-ESO-1 expression in lung cancer cells

Abstract P103 Bonnie Scott . . . . . . . . . . . . . . . . . . . . . . . . .Page 153Evolution of centromere-binding proteins and their interactionswith centromere DNA in Arabidopsis

Abstract P104 David Shechter . . . . . . . . . . . . . . . . . . . . . . .Page 154Histone H2A arginine3 is mono- and symmetrically-dimethylated by a complex of PRMT5 and the WD-repeat proteinMEP50 in Xenopus laevis eggs

Abstract P105 Yoichi Shinkai . . . . . . . . . . . . . . . . . . . . . . . .Page 155H3K9 methylation and germ cell development

Abstract P106 Krishna Sinha . . . . . . . . . . . . . . . . . . . . . . . .Page 156Inhibition of the transcriptional activity of osterix byinteractions with NO66, a jumonji family chromatin protein

Abstract P107 Karen Smith . . . . . . . . . . . . . . . . . . . . . . . . .Page 157Identification and characterization of novel HDAC-associatedproteins that regulate cancer cell growth

Abstract P108 Matthew Smith . . . . . . . . . . . . . . . . . . . . . . .Page 158Chromatin- mediated silencing of immune response genes

Abstract P109 Hae-Ryong Song . . . . . . . . . . . . . . . . . . . . .Page 159Coordination of transcriptional regulation and chromatinmodification of Arabidopsis circadian clock genes

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Poster Index


Abstract P110 Stacey Southall . . . . . . . . . . . . . . . . . . . . . . .Page 160Structural studies of histone methyltransferases

Abstract P111 Maike Stam . . . . . . . . . . . . . . . . . . . . . . . . . .Page 161Molecular analysis of chromatin changes involved in b1paramutation, an allele-dependent transfer of epigeneticinformation

Abstract P112 Sean Taverna . . . . . . . . . . . . . . . . . . . . . . . .Page 162Connecting H3 methylation and acetylation: The role of Yng1 intranscription

Abstract P113 Tage Thorstensen . . . . . . . . . . . . . . . . . . . . .Page 163The Arabidopsis SUVR proteins define a novel subgroup ofSET domain proteins associated with the nucleolus

Abstract P114 Christopher Topp . . . . . . . . . . . . . . . . . . . . .Page 164Unusually-sized centromeric RNAs associate with maizecentromeric chromatin

Abstract P115 Martin Tribus . . . . . . . . . . . . . . . . . . . . . . . . .Page 165Molecular mechanisms of histone variant H3.3 assembly by themotor protein CHD1

Abstract P116 Christopher Vakoc . . . . . . . . . . . . . . . . . . . .Page 166A profile of histone lysine methylation generated bymammalian gene transcription

Abstract P117 Claudius Vincenz . . . . . . . . . . . . . . . . . . . . .Page 167Visualizing polycomb group protein interactions with histonesin vivo

Abstract P118 Vikki Weake . . . . . . . . . . . . . . . . . . . . . . . . .Page 168The SAGA histone acetyltransferase complex functions in thedevelopment of neuronal connectivity in the Drosophilacompound eye

Abstract P119 Stephanie Williams . . . . . . . . . . . . . . . . . . . .Page 169Mechanistic insights into promoter chromatin disassembly

Abstract P120 Jon Wilson . . . . . . . . . . . . . . . . . . . . . . . . . .Page 170Structural studies of SET domain methyltransferases

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Abstract P121 Zhaodong Xu . . . . . . . . . . . . . . . . . . . . . . . .Page 171Remote elements critical for cytokine induced gene expression

Abstract P122 Xiaofang Yang . . . . . . . . . . . . . . . . . . . . . . .Page 172Dissecting SWI/SNF ATP-dependent chromatin remodelingcomplex in Saccharomyces cerevisiae

Abstract P123 Juan I. Young . . . . . . . . . . . . . . . . . . . . . . . .Page 173Post-transcriptional functions of MeCP2

Abstract P124 Veronica Yu . . . . . . . . . . . . . . . . . . . . . . . . . .Page 174Over-expression of Cks proteins causes gene derepression inSaccharomyces cerevisiae

Abstract P125 Rebekah Zinn . . . . . . . . . . . . . . . . . . . . . . . .Page 175hTERT is expressed in cancer despite promoter DNAmethylation by preservation of unmethylated DNA and activechromatin around the transcription start site

Additional poster submissions

Abstract P126 Yoshimitsu Takahashi . . . . . . . . . . . . . . . . . .Page 176Degree of SUMO modification as a differential tag for targetingto specific chromosomal domains

Abstract P127 Marna S. Costanzo . . . . . . . . . . . . . . . . . . . .Page 177The evolutionary conservation of chromatin modifying proteinsin malaria

Abstract P128 Philippe Prochasson . . . . . . . . . . . . . . . . . . .Page 178Functional characterization of the HIR corepressor complex

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Poster Index


Steve Henikoff Abstract 1Epigenetic patterns generated by histone replacement

Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109-1024, U.S.A.

Histone H3 is deposited at replication, but it is replaced at active genes by the constitutivehistone variant, H3.3. We have used chromatin affinity purification of biotin-tagged H3.3 tomap histone replacement throughout the Drosophila genome. Replacement is especiallyprominent at active genes, corresponding to sites of abundant RNA polymerase II andmethylated H3 lysine-4 throughout the genome. Active genes are depleted of histones atpromoters and are enriched in H3.3 from upstream to downstream of transcription units.Histone replacement patterns differ between the dosage compensated X-chromosome andautosomes downstream of gene promoters, suggesting that dosage compensation isachieved by modulating transcriptional elongation. Histone replacement is low overall at theBithorax Complex, but surprisingly, Polycomb Response Elements are sites ofconspicuously high histone turnover, whose peaks precisely correspond to nucleasehypersensitive sites. We also observe high levels of histone turnover at the “poised”promoters of heat shock genes. We propose that the remodeling process responsible forhistone replacement patterns at cis-regulatory elements maintains continuous accessibilityof DNA to trans-acting factors, providing a simple general mechanism for cellular memory.

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Abstracts – Oral


Yang Shi Abstract 2The identification of histone demethylases established thedynamic and reversible nature of histone methylation regulation

In this presentation, I will discuss our continued efforts to catalog histone demethylases andto understand their roles in development and human diseases.

19

Abstracts - Oral


Jesper Christensen Abstract 3The retinoblastoma tumor suppressor binding protein RBP2is a transcriptional repressor demethylating tri- anddimethylated lysine 4 on Histone H3

Jesper Christensen1, Karl Agger1, Paul A. C. Cloos1, Diego Passini1, KlausH. Hansen1 and Kristian Helin1, 2

1Biotech Research & Innovation Centre, Fruebjergvej 3,2100 Copenhagen, Denmark;2Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen,Denmark.

The Retinoblastoma tumor suppressor protein, pRB, is a key regulator of cell-cycleprogression, differentiation and senescence, and is often found deregulated in cancer. Torepress transcription upon cell-cycle exit induced by differentiation or oncogene-inducedsenescence, pRB binds directly to members of the E2F transcription factor family andcellular factors, thereby bridging chromatin modifiers to E2F-regulated genes causingchromatin condensation. Here we show that the Jumonji domain containing protein,Retinoblastoma Binding Protein 2 (RBP2), is a transcriptional co-repressor, which modifieschromatin by demethylating tri- and dimethylated lysine 4 on histone 3 (H3K4), a chromatinmark present on active genes. Ectopic expression of RBP2 in human TIG3 fibroblastsinduced a senescent-like phenotype with reduced H3K4 methylation. Similarly, ectopicexpresion of RBP2 in U2OS cells strongly reduced H3K4 methylation when analyzed byimmunofluorescence. Mutation of the Jumonji domain of RBP2 abolished the demethylationactivity. Furthermore, purified recombinant RBP2 efficiently demethylated tri- or dimethylatedH3K4 in vitro using purified calf thymus histones or HeLa cell nucleosomes as substrate forthe enzyme reactions, while other histone methylation marks at H3K9, H3K27, H3K36 andH4K20 were unaffected. Finally, the enzymatic specificity of RBP2 was confirmed by testingtri-, di-, and monomethylated H3K4 peptides as substrate and subsequent massspectrometry analysis of the reaction products. The biological function of RBP2 is currentlynot fully elucidated. However, considering the pRB binding and the demethylation activity ofRBP2, a role for RBP2 in chromatin demethylation and repression of pRB regulated genesis a possibility and is currently being explored.

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Mischa Machius Abstract 4Structural Basis for CoREST-Dependent Demethylation ofNucleosomes by the Human LSD1 Histone Demethylase

Mischa Machius, Maojun Yang, Christian B. Gocke, Xuelian Luo,Dominika Borek, Diana R. Tomchick, Zbyszek Otwinowski and Hongtao Yu

University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX,75390, U.S.A.

Histone methylation regulates diverse chromatin-templated processes, includingtranscription. Many transcriptional corepressor complexes contain lysine-specificdemethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and di-methylated H3-K4 of nucleosomes. We report the crystal structure of the LSD1-CoRESTcomplex. LSD1-CoREST forms an elongated structure with a long stalk connecting thecatalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 likely recognizes a largesegment of the H3 tail through a deep, negatively charged pocket at the active site and ashallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of theSANT2-DNA interaction diminishes CoRESTdependent demethylation of nucleosomes byLSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding tonucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalentnucleosome-binding mode may apply to other chromatin-modifying enzymes that generallycontain multiple nucleosome-binding modules.

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Abstracts - Oral


Ramin Shiekhattar Abstract 5Functional and biochemical characterization of histonedemethylase complexes

Ramin Shiekhattar

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104

Schizosaccharomyces pombe contains two proteins, SWIRM1 and SWIRM2, with closehomology to human histone H3 lysine 4 demethylase. Both proteins contain the aminooxidase catalytic domain and a recently described DNA interaction SWIRM domain. Ourresults indicate that while SWIRM2 is an essential gene, cells lacking SWIRM1 are viable.We found that SWIRM1 and SWIRM2 are stably associated in a multiprotein complex, butintriguingly, unlike their human counterpart, S. pombe SWIRM complex contains neither ahistone deacetylase (HDAC) nor any detectable demethylase activity. Genome-widechromatin immunoprecipitation unexpectedly showed the absence of both SWIRM proteinsfrom heterochromatic domains. Instead, consistent with biochemical analyses, SWIRM1 andSWIRM2 co-localize to a common set of target gene promoters whose functions areimplicated in diverse processes including mitochondrial metabolism and transcriptionalregulation. Importantly, we show that SWIRM1 is not only required for optimum transcriptionof its target genes but also display a global role in regulation of antisense transcription.

22



Yi Zhang Abstract 6Histone demethylation by the JmjC domain-containingproteins

Yi Zhang

Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill,NC 27599, U.S.A.

Posttranslational histone modifications play an important role in regulating chromatindynamics and function. One of the modifications, methylation, occurs on both lysine andarginine residues and participates in diverse range of biological processes includingheterochromatin formation, X-chromosome inactivation, and transcriptional regulation. Whileacetylation, phosphorylation, and ubiquitylation are dynamically regulated by enzymes thatcatalyze the addition and removal of a particular modification, enzymes that are capable ofremoving methyl groups were not known until recently. Using a novel demethylase assay, wehave identified a family of JmjC domain-containing histone demethylases. The mechanismof demethylation and biological significance of these demethylases will be discussed.

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Abstracts - Oral


Francis Stewart Abstract 7Epigenetic aspects of lineage commitment

Glaser, S., Lubitz, S., Anastassiadis, K., Siebler, J., Schwenk, F. andStewart, A.F.

BioInnovationsZentrum, Technische Universitaet Dresden, Germany; ArtemisPharmaceuticals, Cologne, Germany

In higher eukaryotes, somatic cells differ epigenetically from the pluripotent cells of earlydevelopment. Consequently it is possible that epigenetic mechanisms play important rolesin lineage commitment and cellular differentiation. In mammals, the simplest modelsuggests that the epiblast is pluripotent because its chromatin is epigenetically naive andlineage commitment restricts pluripotency via the imposition of epigenetic marks. A potentialcorollary to this model suggests that cellular identity in the adult is maintained, in part, byepigenetic mechanisms.Recent progress has highlighted the importance of three histone lysine methylations inepigenetics. Whereas methylation of histone 3 lysine 9 (H3 K9) and H3 K27 directinheritable states of gene silencing, methylation of H3 K4 is associated with geneexpression. Mammals have multiple enzymes for each of these methylations, including atleast six for H3 K4. It is therefore possible that different gene expression programs areregulated by different methyltransferases.To explore these issues, we are studying two of the H3 K4 methyltransferases, Mll and Mll2,in mouse development. These two sister genes have arisen by a gene duplication and areclosely related in many ways. However they regulate different genes. Notably Mll regulatesthe Hoxa complex whereas Mll2 the Hoxb complex. Based on experiments with conditionalmutagenesis and studies in utero and in ES cells, we conclude that epigenetic mechanismsare not essential for lineage commitment decisions, rather they contribute to securing andco-ordinating decisions with notable effects on timing and the regulation of apoptosis.

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Henk Stunnenberg Abstract 8Title and abstract unavailable

25

Abstracts - Oral


Ali Shilatifard Abstract 9H2B monoubiquitination and H3K4 methylation via COMPASS

Ali Shilatifard

Saint Louis University Cancer Center, Saint Louis University School of MedicineSaint Louis, MO 63104

Chromosomal rearrangements and translocations play a major role in the pathogenesis ofhematological malignancies. The trithorax related mixed lineage leukemia (MLL) genelocated on chromosome 11q23 is rearranged in a variety of aggressive human B and Tlymphoid tumors as well as acute myeloid leukemia (AML) in both children and adults. Inorder to better define the role of MLL in pathogenesis of leukemia, we have been studyingthe biochemical properties of MLL and MLL-related proteins from several differentorganisms. We have demonstrated that the MLL homologue in yeast, the Set1 protein, existin a macromolecular complex we call COMPASS. COMPASS is a histonemethyltransferases capable of mono- di and trimethylating the fourth lysine of histone H3.Previously, we demonstrated that the ubiquitin-conjugating enzyme Rad6 and its E3 ligaseBre1 and several other factors are required for COMPASS mediated methylation of H3K4through regulation of monoubiquitination of H2B at K123. Here, I will discuss our recentfindings regarding the molecular mechanism and the role of H2B monoubiquitination in theregulation of H3K4 methylation by COMPASS.

26



Paulo Sassone-Corsi Abstract 10A chromatin remodeling clock

Jun Hirayama, Masao Doi, Saurabh Sahar, Benedetto Grimaldi, DavidGauthier, Yasukazu Nakahata and Paolo Sassone-Corsi

Department of Pharmacology, School of Medicine, University of California, Irvine.

Circadian rhythms are the overt consequences of biological clocks, endogenous timersacting within cells. At the molecular level, circadian clocks are constituted by ‘clock genes’,some of which encode proteins able to feedback and inhibit their own transcriptionCircadian rhythms are regulated by clocks located in specific structures of the centralnervous system – such as the suprachiasmatic nucleus (SCN) in mammals – but also byperipheral oscillators present in various other tissues. It is now established that an intrinsiccircadian pacemaker functions in virtually each cell. Importantly, about 15% of all genes areexpressed in a circadian manner. It is thereby conceivable to invoke large-scale events ofchromatin remodeling in order to accommodate these global changes in gene expression.The molecular machinery that governs circadian rhythmicity comprises proteins whoseinterplay generates time-specific transcription of clock genes. The role of chromatinremodeling in a physiological setting such as the circadian clock has been unclear. We haveshown that the protein CLOCK, a central component of the circadian pacemaker, hashistone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme Abinding motifs within the MYST family of HATs. CLOCK displays high sequence similarity toACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity forhistones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HATfunction. The HAT activity of CLOCK is essential to rescue circadian rhythmicity andactivation of clock genes in Clock-mutant cells. Identification of CLOCK as a novel type ofDNA-binding HAT reveals that chromatin remodeling is crucial for the core clock mechanismand identifies unforeseen links between histone acetylation and cellular physiology.

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Abstracts - Oral


Sung Hee Baek Abstract 11A Novel Link between SUMO Modification of a ChromatinRemodeling Complex and Cancer Metastasis

Jung Hwa Kim1,3, Hyejin Nam1,3, Hee June Choi1, Bogyou Kim1, Ji MinLee1, Ik Soo Kim1, Keun Il Kim2, and Sung Hee Baek1

1Department of Biological Sciences, Seoul National University, Seoul 151-746, South Korea,2Department of Biological Sciences, Sookmyung Women's University, Seoul 140-742, SouthKorea

3These authors contributed equally

Defining the functional modules with transcriptional regulatory factors that govern switchingbetween repression and activation events is a central issue in biology. We have reportedthe dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate ametastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumormetastasis, and further which signaling factors confer repressive function on reptin andhence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., NatureCell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealedthe presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptinthat underlies its repressor function. DeSUMOylation of reptin alters the repressive functionof reptin and its association with HDAC1. Further, SUMOylation status of reptin modulatesthe invasive activity in cancer cells with metastatic potential. This provides a clear definitionof the functional model and a novel insight for linking SUMO modification to cancermetastasis. As a follow-up study, we will address novel findings on the function of newlyidentified histone methyltransferase as a component of reptin, linking chromatin remodelingprocess and cancer metastasis.

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Laszlo Tora Abstract 12The simultaneously dimethylated Lys-9 and phosphorylatedSer-10 tails of histone H3 adopt different conformationsduring mitosis

Tora L., Eberlin A., Oulad-Abdelghani M., Robert F., Grauffel C., SpehnerD., Wurtz J-M., Schultz P. and Dejaegere A.

Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGMBC), UMR 7104 CNRS,ULP, INSERM, INSERM U.596, Parc d’Innovation,1, rue Laurent Fries, BP 10142, 67404Illkirch Cedex, C.U. de Strasbourg, France

Eukaryotic cells possess mechanisms for condensing and decondensing chromatin.Chromatin condensation is particularly evident during mitosis and cell death induced byapoptosis, whereas chromatin decondensation is necessary for replication, repair,recombination and transcription. Histones are among the numerous DNA binding proteinsthat control the level of DNA condensation, and post-translational modification of histonetails plays a critical role in the dynamic condensation/decondensation that occur atnumerous cellular processes. Post-translational modifications, alone or in combination, candirect distinct downstream events. The association of lysine (K) 9 dimethylation (di-Me), ahallmark of the heterochromatin, with serine (S) 10 phosphorylation (P), a marker ofmitosis, on the same histone H3 tail, as well as the idea of a structured histone-tail, haslong been controversial. Interestingly, by using a specific antibody, we detect a histone H3tail conformation, which contains simultaneously K9(di-Me) and S10(P) that appears onlybetween the late prophase and the early anaphase steps, being the strongest duringmetaphase. This H3 tail conformation is different from another state, where the K9(di-Me)S10(P) modifications are also simultaneously recognized, but more widely during mitosis.Furthermore, results obtained by confocal and electron microscopy suggest that theconformation of K9(di-Me) and S10(P) histone H3 tails changes during mitosis and canadopt at least two different conformations. This observation has also been confirmed bybiostructural docking and molecular dynamics modelling as well as by competition tests,using various modified peptides. The localisation and the role of these differentconformations in gene regulation and mitotic chromosome condensation will be discussed.

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Abstracts - Oral


Tony Kouzarides Abstract 13Characterisation of novel histone modifications

Claire Pike, Chris Nelson, Paul Hurd, Andy Bannister and Tony Kouzarides

The Gurdon Institute, Cambridge University, Tennis Court Road, Cambridge, U.K.

We are investigating the mechanism of action of several novel modifications on histone H3.We have previously identified an enzyme FPR4 in yeast that can isomerise prolines in the tailof H3. A mammalian enzyme that can accomplish similar functions has been identified and isunder characterisation. In addition, we have identified a new phosphorylation site on humanH3 by mass spectrometry. Specific antibodies raised against this site are now being used toestablish the kinase pathways that mediate this phosphorylation event.

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Thomas Jenuwein Abstract 14Epigenetic control by histone methylation

Thomas Jenuwein

IMP Vienna, Dr. Bohrgasse 7, Austria

Epigenetic mechanisms, such as histone modifications, control eukaryotic developmentbeyond DNA-stored information. We are analyzing histone lysine methylation in mammalianchromatin to further dissect its role(s) in chromosome organization, gene regulation,genome stability and overall epigenetic control. While there is under-representation ofrepressive histone marks in quiescent (resting), stem and regenerating cells, there is aselective accumulation of aberrant histone lysine methylation profiles in aging, ‘stressed’and tumor cells. We have generated mutant mice that lack crucial HMTases, such as theSuv4-20h enzymes. In these Suv4-20h double-null mice, there is a genome-wide transitionfrom H4K20 tri- to H4K20 mono-methylation, which appears to impair stress-induced andprogramd DNA damage response. In addition, we have screened chemical libraries (incollaboration with Boehringer Ingelheim, Ridgefield U.S.A.) and identified a small moleculeinhibitor for the G9a HMTase. This novel compound, BIX-01294, is the first HMTase inhibitorthat can be used to transiently modulate H3K9me2 levels in mammalian chromatin. Finally,we have been characterizing jumonjiC-containing proteins that represent hydroxylases withthe potential to remove repressive H3K9me3 marks. Together, these approaches promise toyield new insights into the plasticity of cell fate decisions and may offer novel strategies torevert aberrant development.

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Abstracts - Oral


Roberta Benetti Abstract 15The role of Dicer in the regulation of chromatin at telomeres

Roberta Benetti1*, Susana Gonzalo1,4*, Stefan Schoeftner1, Isabel Jaco1,Purificacion Muntildedoz1, Elizabeth Murchison2, Thomas Andl3, PeterKlatt1, Sarah Millar3, Gregory Hannon2 and Maria A. Blasco1

1Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National CancerCentre (CNIO), Madrid E-28029, SPAIN; 2Cold Spring Harbor Laboratory, NY 11724, U.S.A.;3Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104-6100,U.S.A.; 4Radiation and Cancer Biology Division, Department of Radiation Oncology,Washington University School of Medicine, St. Louis, MO 63108, U.S.A.

Dicer has been proposed to have a role in the maintenance of silencing at centromeres inseveral organisms, including mammals. Here we describe a role for Dicer in regulatingmammalian telomeric chromatin. In particular, mouse ES cells and skin keratinocytesconditionally deleted for Dicer show aberrantly elongated telomeres compared to wild-typecontrols, concomitant with increased telomeric recombination. This occurs in the absence ofchanges in TRF1 and TRF2 expression and with decreased telomerase activity in Dicer-nullcells. The long-telomere phenotype of Dicer-null cells is accompanied by an increaseddensity of histone heterochromatic marks at telomeric chromatin, such as histone 3 lysine 9(H3K9) and histone 4 lysine 20 (H4K20) tri-methylation, and by decreased histoneacetylation, supporting the idea of a silencing of telomeric chromatin in the absence ofDicer. In support of this, we observed a decreased abundance of telomeric RNA transcriptsin Dicer-null cells. All together, these results demonstrate an unprecedented role for Dicer inthe regulation of mammalian telomeric chromatin. Dicer has been proposed to have a rolein the maintenance of silencing at centromeres in several organisms, including mammals.Here we describe a role for Dicer in regulating mammalian telomeric chromatin. Inparticular, mouse ES cells and skin keratinocytes conditionally deleted for Dicer showaberrantly elongated telomeres compared to wild-type controls, concomitant with increasedtelomeric recombination. This occurs in the absence of changes in TRF1 and TRF2expression and with decreased telomerase activity in Dicer-null cells. The long-telomerephenotype of Dicer-null cells is accompanied by an increased density of histoneheterochromatic marks at telomeric chromatin, such as histone 3 lysine 9 (H3K9) andhistone 4 lysine 20 (H4K20) tri-methylation, and by decreased histone acetylation,supporting the idea of a silencing of telomeric chromatin in the absence of Dicer. In supportof this, we observed a decreased abundance of telomeric RNA transcripts in Dicer-nullcells. All together, these results demonstrate an unprecedented role for Dicer in theregulation of mammalian telomeric chromatin

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Mareike Puschendorf Abstract 16Ezh2 independent targeting of PRC1 proteins to paternalconstitutive heterochromatin in mouse pre-implantationembryos

Mareike Puschendorf

Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4057 Basel Switzerland

In mammals, fertilization triggers a cascade of events leading to the formation of atotipotent embryo from two highly specialized gametes. During this process both parentalgenomes undergo major epigenetic reprogramming, suggesting a potential causalrelationship between the two events. Several immunofluorescence studies indicate thatchromatin states of maternal and paternal genomes are initially highly asymmetric.Whereas the maternal genome inherits many distinct types of histone lysine methylation,the paternal genome is de novo methylated at different lysine residues in a highly spatiallyand temporally coordinated manner after the protamine to histone exchange. At thematernal genome, constitutive heterochromatin is labeled by modifications characteristic ofthe Suv39h pathway (such as H3K9 and H4K20 tri-methylation and binding of HP1b).Importantly, in proliferating somatic cells Suv39h function is required to maintain mitoticgenome stability. Surprisingly, paternal constitutive heterochromatin in early embryos isdevoid of the canonical Suv39h-dependent chromatin marks. Instead, we observe thatvarious proteins of the Polycomb Repressive Complex 1 (PRC1) are targeted to constitutiveheterochromatin of only the paternal genome. By using embryos maternally and zygoticallydeficient for Ezh2, we demonstrate that the parental-origin-specific labeling is independentof Ezh2 function and H3K27 tri-methylation. PRC1 binding to paternal heterochromatin isstably transmitted over several mitotic divisions suggesting the existence of a memory ofparental identity of constitutive heterochromatin in pre-implantation embryos.

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Abstracts - Oral


Edith Heard Abstract 17The nuclear and epigenetic dynamics of X-chromosomeinactivation in the mouse

Edith Heard

Mammalian Developmental Epigenetics Group, CNRS UMR 218 - Nuclear Dynamics andGenome Plasticity Curie Institute, 26 rue d’Ulm, 75248 Paris Cedex 05, France

In female mammals, one of the two X chromosomes is converted from the active euchromaticstate into inactive heterochromatin during early embryonic development. This process, knownas X-chromosome inactivation, results in the transcriptional silencing of over a thousandgenes and ensures dosage compensation between the sexes. X inactivation is a dramaticexample of mammalian epigenetics, involving differential regulation of two homologouschromosomes within the same nucleus, in a mitotically heritable but developmentallyreversible manner. We are interested in the mechanisms and kinetics of this process in earlymouse embryos and differentiating embryonic stem (ES) cells. X inactivation is a highlydynamic process during early development (Okamoto et al, 2004) and we are interested indefining the epigenetic marks that underlie its initiation and its maintenance. Given the mono-allelic character of X inactivation, we are also investigating the role of sub-nuclear compart-mentalization in this process, both at the level of the master control locus of X inactivation,the Xic, and the non-coding Xist transcript it produces, that is responsible for inducingtranscriptional silencing in cis. In this context, we have recently discovered that the two Xicstransiently co-localise just prior to random monoallelic up-regulation of Xist and the onset ofX inactivation (Bacher et al, 2006). This co-localisation seems to be important for ensuringthat X inactivation is triggered when more than one Xic is present. Recent evidence will bepresented for a new region of the Xic that seems to be critical for bringing the two loci togetherin trans and that is characterized by specific histone modifications.

Refs:Okamoto,I., Otte,A., Allis,C.D., Reinberg,D. and Heard,E. (2004) Epigenetic dynamics ofimprinted X inactivation during early mouse development. Science, 303, 644-649Bacher,C., Guggiari,M., Brors,B., Augui,S., Avner,P., Eils,R. and Heard,E. (2006) Transientcolocalization of X-inactivation centres accompanies the initiation of X inactivation. NatureCell Biology, 8, 293-239.

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Adrian Bird Abstract 18MeCP2: molecular interactions and phenotypic stability in amouse model of Rett Syndrome

Jacky Guy1, Xinsheng Nan1, Jianghui Hou2, Skirmantas Kriaucionis1 andAdrian Bird1

1Wellcome Trust Centre for Cell Biology, The University of Edinburgh, The Kings Buildings,Edinburgh EH9 3JR, U.K., 2Molecular Medicine Centre, The University of Edinburgh,Western General Hospital, Edinburgh EH4 2XU, U.K.

Rett Syndrome (RTT) is a profound neurological disorder that almost exclusively affectsgirls. More than 80% of patients carry a new mutation in one copy of the X-linked MECP2gene and this is now established as the primary cause of the condition. Overt symptomsshow delayed onset in girls between 6 and 18 months of age and include developmentaldelay, loss of purposeful limb use and breathing abnormalities. As there is no obviousneurodegeneration in post-mortem brains of RTT patients, the question of reversibilityarises and is of obvious relevance for therapeutic approaches to RTT. We earlier created amouse model for RTT that lacks an intact Mecp2 gene and mimicks several features of thedisorder including late inset. Using a mouse with an Mecp2 allele that can be conditionallyactivated, we are asking whether neuronal defects in the young adult can be rectified ifMeCP2 is provided after abnormal neuronal morphology and symptoms have arisen. Canswitching on MeCP2 in these animals reverse the phenotype, or is it too late? In addition tothese physiological studies, we have identified the Swi/Snf motor protein ATRX as anMeCP2 binding partner. Mutations in both MECP2 and ATRX genes cause X-linked mentalretardation and we have preliminary evidence for interdependence in the mouse brain.

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Abstracts - Oral


Jon Penterman Abstract 19DNA demethylation in Arabidopsis thaliana

Jon Penterman1, Daniel Zilberman2, Jin Hoe Huh1, Tracy Ballinger2,3,Steven Henikoff2,3, Robert Fischer1

1Department of Plant and Microbial Biology, University of California, Berkeley, California94720, U.S.A. 2Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle,Washington, 98109, U.S.A. 3Howard Hughes Medical Institute

Cytosine DNA methylation is an epigenetic modification that functions in a number ofprocesses, one of which is genome defense against transposons and repetitive elements. InArabidopsis thaliana DNA is methylated by methyltransferases whose specificity isdetermined by parental methylation patterns, histone modifications, and/or small RNAs.Here we show that methylation at many loci throughout the genome is actively removed bya DNA demethylation pathway. The DEMETER-LIKE (DML) DNA glycosylases, which excise5-methylcytosine and initiate the base excision DNA repair pathway, mediate this process.Using genome-tiling arrays, we detected nearly two hundred discrete loci that aredemethylated in a DML-dependent manner. We find that DML demethylation primarilyoccurs at the 5’ and 3’ ends of genes, a pattern opposite to the overall distribution of wild-type DNA methylation. Our results show that DML-dependent DNA demethylation is afundamental pathway that edits the Arabidopsis methylation profile. We believe that DMLdemethylation provides a protective buffer against the methylation pathway of Arabidopsis,which might indirectly enable Arabidopsis to have a robust defense pathway for repressingtransposons and repetitive elements.

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Francois Fuks Abstract 20The Polycomb Group protein EZH2 is recruited to promotersby MECP2

Emmanuelle Vire1, Helene Denis1, Esteban Ballestar2, Yvan de Launoit3,Manel Esteller2 and Francois Fuks1

1Free University of Brussels, Faculty of Medicine, Laboratory of Cancer Epigenetics, 808route de Lennik, 1070 Brussels, Belgium; 2CNIO, Cancer Epigenetics Group, C/ MelchorFernandez Almagro 3,28029-Madrid, Spain; 3Institut de Biologie de Lille, 1 rue Calmette,59021 Lille, Cedex, France

Polycomb Group (PcG) proteins and DNA methylation are fundamental epigenetic systemsinvolved in gene silencing. Recently we have uncovered a close connection between thesetwo systems: the PcG protein EZH2 can control DNA methylation (1).Here we show that conversely, CpG methylation can influence EZH2 function through themethyl-CpG-binding protein MECP2. We demonstrate that EZH2 interacts physically withMECP2 in vivo. Chromatin immunoprecipitations indicate that the presence of MECP2 isrequired for binding of EZH2 to target promoters. Genome-wide location analysis withantibodies against these proteins are under way to explore whether there is a cross-talkbetween EZH2 and MECP2 across promoters within the genome.Our results suggest that MECP2 may act as a molecular scout for PcG recruitment tochromatin. They could shed light on the poorly understood mechanisms by whichmammalian Polycomb Group proteins are targeted to promoters.

Ref.(1) Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A,Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F.Nature. 2006 Feb 16:871-4.

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Shelley Berger Abstract 21

Factor and histone covalent modifications in genome regulation

Shelley L. Berger

The Wistar Institute, Philadelphia, PA, 19104, U.S.A.

Genomic structure and function is regulated in part through covalent post-translationalmodifications (PTMs) of factors and histones, including acetylation (ac), methylation (me),phosphorylation (ph), ubiquitylation (ub), and sumoylation (su). There are an enormousnumber of factor and histone PTMs. To make sense of this bewildering complexity, wefocuses on patterns, temporal sequences, and cross-talk between PTMs in the yeast S.cerevisiae and mammalian cells.In mammals we study PTMs of DNA-bound transcription factors, using the tumorsuppressor and transcription factor p53 as a model. We currently focus on methylation bythe SET domain methyltransferase Smyd2. Smyd2 methylates p53 at K370, adjacent to thepreviously identified methylation site at K372. K372me is associated with transcriptionalactivation by p53, and inhibits K370me, which in contrast, is associated with transcriptionalrepression by p53. Thus, there is regulatory cross-talk between activating and repressinglysine methylation. Our most recent results indicate that demethylation occurs within p53.A methylation/demethylation pathway will be discussed in detail.

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Danny Reinberg Abstract 22A molecular understanding of epigenetics

Danny Reinberg

NYU-School of Medicine, NY, U.S.A.

Epigenetics encompasses changes in gene expression profiles that occur withoutalterations in the genomic DNA sequence of a cell. This arises from the dynamic processesthat structure regions of chromosomal DNA through a range of compaction in eukaryotes.The altered pattern of gene expression is pivotal to cellular differentiation and developmentand is inherited by daughter cells thereby maintaining the integrity, specifications, andfunctions for a given cell type. Aberrancies in this epigenetic process give rise toperturbations that are also inherited and disruptive to normal cellular properties. The histoneproteins that package DNA into chromatin are subject to post-translational modificationsgenerating different chromatin structures. While euchromatin has a relaxed structurepermissive to transcription, constitutive heterochromatin is densely packed and inaccessibleto transcription factors. On the other hand, facultative heterochromatin is repressive, but canbe altered in its properties to become active. Our goals are to identify the molecularmechanisms controlling the formation of facultative heterochromatin and the epigeneticparameters that ensure its propagation through cell divisions.

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Jessica Tyler Abstract 23The mechanistic basis for the requirement of promoterchromatin disassembly for transcriptional activation

Jessica K Tyler1, Stephanie Williams1, Melissa Adkins1, Christine English1

and Mair Churchill2

1 Department of Biochemistry and Molecular Genetics, 2Department of Pharmacology,University of Colorado at Denver and Health Sciences Center, Aurora CO 80045

Nucleosomes appear to be disassembled from the promoters of transcriptionally activegenes in Eukaryotic species spanning from yeast to humans. We have previously shownthat this promoter chromatin disassembly is essential for transcriptional activation of thebudding yeast PHO5 and PHO8 genes. Promoter chromatin disassembly is mediated bythe highly conserved histone H3/H4 chaperone Anti-silencing function 1 (Asf1) duringactivation of the PHO5 and PHO8 genes upon phosphate depletion and during activation ofthe ADY2 and ADH2 genes upon glucose removal. Using PHO5 as our model system tolearn how promoter chromatin disassembly activates gene expression, we have discoveredthat Asf1-mediated promoter chromatin disassembly is required for recruitment of TBP andRNA polymerase II, but not for recruitment of the Pho4 and Pho2 activators. Furthermore,accumulation of SWI/SNF and SAGA at PHO5 required promoter chromatin disassembly.We have also uncovered a novel requirement for SWI/SNF and SAGA in chromatindisassembly to facilitate activator recruitment to the nucleosome-buried binding site in thePHO5 promoter that is distinct from the stable recruitment of SWI/SNF and SAGA afterchromatin disassembly.Towards addressing the mechanism whereby Asf1 mediates chromatin disassembly, wehave solved the crystal structure of Asf1 in complex with histones H3/H4 to 1.7 Angstromresolution; this is the first time histone proteins have been “seen” outside of the nucleosomeor octamer structures (see Mair Churchill’s abstract for more details). Using mutantsdesigned from the Asf1-H3/H4 structure, we show that Asf1 needs to bind to the H3:H3dimerization surface of the H3/H4 heterodimer in order to achieve chromatin disassemblyfrom the PHO5 promoter. Furthermore, binding of Asf1 to the C-terminal beta strand ofhistone H4, which in itself induces an 180˚ flipping-out of this region of H4 as compared tothe nucleosome, is also required for chromatin disassembly by Asf1. From these results wepropose a “strand capture” model for chromatin disassembly.

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Gratien Prefontaine Abstract 24Epigenetic mechanisms influencing pituitary geneexpression.

Prefontaine G.G., Lunyak, V. and Rosenfeld M.G.

Department of Medicine, University of California, San Diego, 9500 Gilman Drive, CMM-West #345, La Jolla, CA, 92093-0648

During development, tissue-specific transcription factors direct covalent modifications ofchromatin, laying down the foundation for regulated gene transcription. These factors actthrough cis-acting elements located in promoter proximal and distal elements. The pituitarygland provides an excellent model system for studying epigenetic changes in generegulation. Common pluripotent primordial ectodermal cells differentiate to produce apituitary gland composed of 5 major cell types characterized by types of hormone theyproduce and secrete, including: lactotropes, somatotropes, thyrotropes, corticotropes andgonadotropes. I have performed a comprehensive analysis of the CpG DNA methylationstatus of the growth hormone (GH) promoter in mouse pituitary. Early in development theembyonic murine GH promoter was largely CpG methylated. Later postnatally, the GHpromoter was demethylated in a subset of cell types. I have linked the DNA demethylationof the promoter, genetically and spatially with the occupancy of a cell-type specific factor.Furthermore, using a combination of techniques, I show the CpG methylation status of thepromoter associated with specific histone modifications. These results have allowed us topropose a model where the GH gene is differentially repressed by polycomb (long termsilencing) and HP1 (short term repression) proteins in distinct pituitary cell types. UsingBAC recombination to substitute a fluorescent gene into the GH gene locus and to deleteupstream distally located regulatory elements, I have created multiple transgenic mice thatdemonstrate the importance of a GH enhancer or locus control region and a repetitiveelement that are critical for proper regulation of the GH locus.

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Bob Kingston Abstract 25Possible roles in silencing for piRNAs

Bob Kingston, Anita Seto, Nelson Lau, Jinkuk Kim, David Bartel,Jonathan Dennis and Caroline Woo

Massachusetts General Hospital, 185 Cambridge Street, CPZN7250, Boston MA 02114, U.S.A.

Maintaining a silent state requires the targeting of epigenetic regulatory complexes tospecific genes. Small noncoding RNAs have been proposed to play a role in this targeting,based on studies in numerous model organisms. Because these RNAs appeared to beenriched meiotic cells in model organsims, we made extracts from Rat testes to try toidentify RNAs that might be involved in silencing in mammals. From these extracts, wepurified a mammalian complex that might function in transcriptional gene silencing (TGS).This complex, called piRC, contains small RNAs and Riwi, the rat homolog to human Piwi.The RNAs, frequently 29–30 nt in length, are called Piwi-interacting RNAs (piRNAs), 94% ofwhich map to 100 small (<100 kb) genomic loci. Within these loci, the piRNAs distributeacross only one genomic strand, or distribute on two genomic strands but in a divergent,non-overlapping manner. Preparations of piRC contain rRecQ1, which is homologous toqde-3 from Neurospora, a gene implicated in silencing pathways. Recombinant RecQ1 andPiwi family proteins appear to interact directly. Piwi has been genetically linked to TGS inflies, and the purified complex has piRNA-directed slicer activity. These results areconsistent with a gene silencing role for piRC in mammals.

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Michael Grunstein Abstract 26Deacetylation of histone H4 K16 regulates gene activity inyeast

Wei Xie, Amy Wang and Michael Grunstein

Department of Biological Chemistry and the Molecular Biology Institute, Boyer Hall, UCLA,Los Angeles, CA. 90095, U.S.A.

We have found that of all the H4 sites of acetylation, K16 when deacetylated is uniquelyinvolved in the silencing of heterochromatin through its interaction with the silencing proteinSir3. This site is also uniquely involved in gene activation in euchromatin. Surprisingly, whilehistone hyperacetylation is generally correlated with gene activity, it is the hypoacetylation ofK16 that is associated with gene activity genome wide. This occurs in part through itsrecruitment of the bromodomain containing transcription factor, Bdf1. We describe here theenzymes which deacetylate and acetylate K16 and their recruitment during gene activity todynamically regulate transcription.

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Ann Ehrenhofer-Murray Abstract 27A role for the HDAC Rpd3 in establishing eurchromatin-heterochromatin boundaries at yeast telomeres

Stefan Ehrentraut and Ann E. Ehrenhofer-Murray

Universität Duisburg-Essen, 45117 Essen, Germany

Eukaryotic genomes are organized into euchromatic and heterochromatic regions. Cellsneed to ensure that the respective chromatin states are restricted to their location in orderto prevent inappropriate gene expression.In Saccharomyces cerevisiae, spreading of the telomeric Sir2/ Sir3/ Sir4 heterochromatincomplex is prevented by the activity of the HAT complex SAS-I, which acetylates lysine 16of histone H4, a residue that is required in the deacetylated state for the SIR complex tobind to chromatin. In sas2-delete(D) cells, SIR spreads to more centromere-proximalpositions and causes repression of subtelomeric genes. However, the SAS2 deletion is notlethal in yeast.Here, we performed a genetic screen to identify factors that become lethal in the absence ofSas2. Surprisingly, we found that the absence of the HDAC Rpd3 was synthetically lethal incombination with sas2D. The lethality was specific for rpd3D and sas2D in that no otherHDAC deletion was lethal with sas2D, and no other HAT deletion was lethal with rpd3D.Furthermore, the lethality depended on the components of the Rpd3(L) complex and on allSAS-I components. Our observations suggest parallel functions of the two proteincomplexes despite their opposing enzymatic activities.Significantly, we found that the lethality of sas2D rpd3D cells was caused by inappropriatespreading of SIR complexes, because the lethality was suppressed by sir2/3/4D. In line withthis, we found by ChIP analysis that Sir2 was more abundant at telomeres and insubtelomeric regions in rpd3D cells than in wild-type. Furthermore, subtelomeric geneswere more repressed in rpd3D cells than in wild-type as measured by qRT-PCR, and therepression was abrogated in the absence of SIRs.Altogether, our data show a novel and unexpected role for Rpd3 in preventing spreading ofSIR complexes into euchromatic regions, indicating that Rpd3 exerted a boundary functionat yeast telomeres.

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Wyatt Yue Abstract 28CARM1 and Histone Methylation - a Structural Study

W.W. Yue1, V. Thompson Vale1, M. Hassler1, S. Kisakye-Nambozo1,M. Roe1, T. Kouzarides2 and L.H. Pearl1

1Section of Structural Biology, Institute of Cancer Research, 237 Fulham Road, LondonSW3 6JB, UK; 2Gurdon Institute and Department of Pathology, Tennis Court Road,Cambridge CB2 1QN, U.K.

Covalent histone modifications have important roles in transcriptional regulation by effectingchanges in the chromatin structure. Coactivator-associated arginine methyltransferase 1(CARM1) methylates histone H3 at Arg17 and Arg26 upon hormonal activation of nuclearreceptors. It enhances transcriptional activation through synergistic interactions withcoactivators such as GRIP-1 and the histone acetyltransferase CBP.CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic domainflanked by unique N- and C-terminal extensions. We have determined the 2.8 Å structuresof the CARM1 catalytic domain alone, and in complex with the cofactor product AdoHcy.The catalytic domain consists of a cofactor binding region and a nine-stranded ß-barrel.The binding of AdoHcy allows the ordering of the N-terminal helix α1, which formsextensive contacts with AdoHcy. This almost completely buries the cofactor and limits theaccessibility of the arginine substrate to a narrow channel. Helix α1 also forms the upperridge of a proposed substrate binding groove lining the entrance to the active site. Thebottom ridge of this groove is formed by the first 10 residues of the CARM1 C-terminalextension, which is not present in other PRMT structures.Using in vitro methylation and pull-down assays, we have demonstrated that the N- and C-terminal extensions are important in contributing to the enzyme activity of CARM1. Inaddition, CARM1 activity towards histone H3 Arg17 is potentiated by pre-acetylation atLys18, suggesting a possible cross-talk mechanism between histone acetylation andmethylation. We are in the process of determining the structure of a CARM1-cofactor-substrate complex, in order to establish the molecular basis for the substrate specificity ofCARM1 towards histone H3, which is unique among PRMTs.

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Sharon Dent Abstract 29Common and unique factors regulate Set1-mediatedmethylation of the Dam1 kinetochore protein and histone H3

Sharon Y.R. Dent, John A. Latham and Ke Zhang

Univ. Texas M.D. Anderson Cancer Center, Dept. Biochemistry and Molecular Biol., Unit1000, 1515 Holcombe Blvd, Houston, Texas 77030, U.S.A.

We reported last year that deletion of the SET1 methyltransferase gene suppresses defectsin chromosome segregation caused by mutations in the IPL1 aurora kinase in yeast (Zhanget al. Cell 122, 2005). Mutations in other components of the COMPASS complex alsosuppress the ipl1-2 mutation, but mutations in PAF1 or H2B K123 do not. These resultsindicate that Set1 and the COMPASS complex normally oppose functions of Ipl1, but thatthese effects are independent of the functions of Set1 in transcription initiation andelongation that are mediated by Paf1 and H2B ubiquitylation. Moreover, we determinedipl1-2 suppression is not related to loss of H3 K4 methylation in set1 mutant cells. Rather,Set1 is required for methylation of a kinetochore protein, Dam1, and Dam1 methylation atK233 limits phosphorylation of neighboring serines. We have now defined the effects ofmutations in individual COMPASS components on Dam1 methylation in vivo. We have alsobegun to define upstream factors that are required for methylation of both H3 K4 andDam1, and as well as unique factors required for methylation of each of these substrates.Our findings demonstrate that Set1 has important functions in mitosis, and they suggestthat antagonism between lysine methylation and serine phosphorylation is a fundamentalmechanism for controlling protein function.

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Geneviève Almouzni Abstract 30Chromatin assembly factors, histone H3 variants and cell cycle

Dominique Ray-Gallet, Sophie Polo, Jean-Pierre Quivy, Anja Groth,Danièle Roche and Geneviève Almouzni

UMR 218 CNRS, Institut Curie – Recherche, 26 rue d’Ulm, F-75248 Paris cedex 05, France

The ordered assembly of chromatin produces a nucleoprotein template capable ofregulating the expression and maintenance of the genome functions.Factors have been isolated from cell extracts that stimulate early steps in chromatinassembly in vitro. One such factor, chromatin assembly factor-1 (CAF-1), facilitatesnucleosome formation coupled to DNA synthesis. It is thought to participate in a markingsystem at the crossroads of DNA replication and repair to monitor genome integrity and todefine particular epigenetic states. We have begun to approach its critical importance duringearly development in Xenopus laevis and using mammalian cell systems. In addition, wehave now identified a chromatin assembly pathway independent of DNA synthesis. TheHIRA protein appears critical for this pathway in Xenopus egg extracts. Notably, CAF-1 waspart of the the histone H3 complex, H3.1 complex (replicative form) and HIRA of the H3.3complex (replacement form) (Tagami et al, 2004, Nakatani et al, 2004). A major goal in ourlaboratory is now to better integrate the function of these factors in vivo during developmentand also in connection with replication, repair and control of histone pools.We will discuss our recent findings on this topic and the interrelationships with otherassembly factors.

Refs.Groth A., Ray-Gallet D., Quivy J.P., Lukas J., Bartek J. & Almouzni G. (2005) Human Asf1regulates the flow of S-phase histones during replicational stress. Mol. Cell, 17, 301-311.Polo S. & Almouzni G. (2006) Chromatin assembly : a basic recipe with various flavors.Current Opinion in Genetics and Development, 16, 104-111.Gérard A., Koundrioukoff S., Ramillon V., Sergère J.C., Mailand N., Quivy J.P. & Almouzni G.(2006) The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit ofChromatin Assembly Factor 1 with proliferating cell nuclear antigen. EMBO Reports , 7, 817-823.Polo S., Roche D. & Almouzni G. (2006) Evidence for new histone incorporation markingsites of UV-repair in human cells. Cell (in press).Loyola A., Bonaldi T., Roche D., Imhof A. & Almouzni G. (2006) Modifications on histone H3variants before chromatin assembly potentiate their final epigenetic state. Mol. Cell (in press).

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Dmitry Fyodorov Abstract 31ATP-dependent deposition of Histone H3.3 by DrosophilaCHD1 in vivo

Alexander Konev1, Martin Tribus2, Alexandra Lusser2 and Dmitry Fyodorov1

1Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A.; 2Innsbruck MedicalUniversity, Innsbruck, A-6020, Austria.

The assembly of nucleosomes in vitro is mediated by the concerted action of two groups offactors – histone chaperones, such as CAF-1, NAP-1, Asf1 and HIRA, and ATP-utilizingenzymes, such as ACF/CHRAC, RSF and CHD1. However, the respective roles of thesefactors in the chromatin assembly process in vivo remain controversial. For instance, it ispossible that ATP-dependent factors are dispensable for the histone deposition andparticipate only in the spacing of the nascent nucleosomes.Drosophila CHD1, in conjunction with the histone chaperone NAP-1, can mediate theassembly of periodic arrays of nucleosomes in vitro. To elucidate the biological function ofCHD1 we generated mutant alleles of Chd1. Homozygous null Chd1 female flies survive toadulthood and lay fertilized eggs, but the embryos die before hatching. We have analyzedthe chromosome structure in developing Chd1 embryos and found that the absence ofCHD1 impedes decondensation of paternal sperm chromatin and consequently results inthe development of haploid embryos.Sperm decondensation is the earliest developmental instance of genome-scale chromatinassembly. The male pronucleus undergoes profound chromatin reorganization, as paternalprotamines are replaced with maternal histones. This process occurs in a replication-independent manner and involves H3.3 variant but not the canonical H3. We found that inthe absence of maternal CHD1, H3.3 fails to become incorporated into male chromatin.Thus, we demonstrate that CHD1 directly mediates the loading of H3.3 onto DNA in vivo.The function of CHD1 in histone H3.3 deposition, combined with the recent finding that thedelivery of H3.3 to the male pronucleus is dependent on the chaperone HIRA, support amolecular model in which CHD1 utilizes HIRA-delivered histones to assemble H3.3-containing nucleosomes. The combined action of CHD1 and HIRA defines a novel pathwayfor the replication-independent deposition of variant histones into chromatin.The nucleosome assembly in the male pronucleus takes place in the transcriptionally silentphase of Drosophila embryonic development. However, both H3.3 and CHD1 are expectedto function in transcription-coupled nucleosome assembly. Therefore, we analyzed H3.3incorporation into chromatin during later developmental stages, after the onset of zygotictranscription. We discovered that elimination of maternal CHD1 blocks the assembly ofH3.3-containing nucleosomes in transcriptionally active chromatin.Our work provides the first conclusive evidence that ATP-dependent mechanisms areutilized for histone deposition during chromatin assembly in vivo. Hence, molecular motorproteins, such as CHD1, function not only in remodeling of existing nucleosomes but also inde novo nucleosome assembly from DNA and histones.

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Roberto Mantovani Abstract 32The histone-fold trimer NF-Y is required to define positivehistone marks in CCAAT-promoters: a genome-wide analysis.

Michele Ceribelli, Giacomo Donati, Diletta Dolfini, Giulio Pavesi,Alesaandra Viganò and Roberto Mantovani

Dipartimento di Scienze Biomolecolari, U. di Milano, Via Celoria 26 20133 Milano, Italy

The CCAAT box is a promoter element, bound by the NF-Y trimer, composed of an H2A-H2B-like dimer and a sequence-specific subunit, NF-YA. To gain an unbiased view of NF-Ybinding in vivo, we performed ChIP on chips with anti-NF-YB antibodies on 3 platforms. (i)CpG islands arrays identified 300 genes targeted by NF-Y. Surprisingly, 41% of NF-Y sitesare not in promoters, but in introns or at distant 3’ or 5’ loci (1). (ii) A more sensitive oligo-based chip containing 179 human promoters indicated that NF-Y binds to 40/54% ofpromoters, essentially all containing one -or more- CCAAT boxes (2). (iii) We performedlocation analysis on the Nimblegen tiling arrays of chromosomes 20/21/22. This methodproved to be highly specific, as none of 24 negative locations scored positive, but missedsome 30% of sites. Consistent with the CpG array, only a minority of the sites -15/20%- arein promoters. A positive correlation with active histone –H3-Acetylation and H3-K4-trimethylation- was established in parallel ChIP on Chip experiments on the same platform.ChIP analysis of cells infected with a dominant negative NF-YA expressing Adenovirusshowed a remarkable decrease in NF-YB local promoter binding and in the abovementioned histone modifications, as well as H3-K79-dimethylation. Similar results wereobtained transfecting cells with siRNA for NF-YB. Consequently, elimination of NF-Y bindingleads to transcriptional impairment. These data establish NF-Y as a crucial factor in theformation of a positive epigenetic environment around the transcription start sites.

Refs1) Testa et al. J. Biol Chem. 280, 13606-13615 (2005).2) Cerebelli et al. Cell Cycle, In press (2006).

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Jerry Workman Abstract 33Histone modification and chromatin remodeling in transcription

Bing Li, Kenneth Lee, Mark Chandy, Michael Carey and Jerry Workman

Stowers Institute for Medical Research 1000 East 50th Street Kansas City, MO 64110 U.S.A.

Nucleosome remodeling and histone modification play crucial roles in the process of genetranscription. Sequence-specific DNA binding transcription activators recruit the SAGAhistone acetyltransferase complex to gene promoters during activation. Targeted acetylationof promoter nucleosomes by the SAGA complex marks them for subsequent displacementby the Swi/Snf nucleoosome remodeling complex. This generates nucleosome free regionswhere the general transcription factors and RNA polymerase II can form a preinitiationcomplex. Following initiation the elongating RNA polymerase associates withacetyltransferases that co-transcriptionally acetylate nucleosomes in the coding region. Thisacetylation is recognize by the bromo-domain containing RSC complex which remodelsnucleosomes to assist polymerase passage. The Set2 histone methyltransferase alsotravels with the polymerase and co-transcriptionally methylates histone H3. This methylationis subsequently recognized by the chromodomain containing Rpd3S histone deacetylasecomplex which removes the co-transcriptional histone acetylation marks returning thestability of nucleosomes within the coding region.

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Karl Agger Abstract P1The role of the polycomb group protein RYBP in oncogeneinduced senescence

Karl Agger1, Paul Cloos1, Michael Lees1 and Kristian Helin1,2

1Biotech Research & Innovation Centre, Fruebjergvej 3 Copenhagen, Denmark;2Faculaty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200Copenhagen, Denmark

Several links between deregulated chromatin modifying activities and cancer have beendescribed. In our laboratory special attention has been applied to the role of PcG genes inthe regulation of cell cycle progression and cancer because of their well- establishedoncogenic potential. In an attempt to identify additional proteins involved in PcG relatedchromatin modification we performed multiple Yeast two hybrid (YTH) screens using PcGgenes as baits. From the results of these screens we constructed a protein interaction mapof the human Polycomb group proteins. We chose to focus on one of the identifiedinteractions between the PcG gene RYBP and the HMT SUV4-20H1. This interaction isinteresting because it links a new repressive enzymatic activity to the PcG protein family. Tofurther characterise the interaction we confirmed the binding between the two proteins byco-immunoprecipitation. We found that RYBP and SUV4-20H1 over-expression inducedheterochromatin foci in U2OS cells. These foci resemble Senescence associatedheterochromatin foci (SAHF). SAHFs are dapi dense heterochromatin foci that appears incells undergoing senescence, a proliferative arrest that provides a barrier to malignanttransformation and contributes to the antitumor activity of certain chemotherapies. We foundthat both RYBP and SUV4-20H1 can induce senescence in diploid fibroblasts upon over-expression. Additionally we found that ectopic RYBP activity can induce SAHF-likestructures and the protein RYBP co-localise with these. Hence, RYBP could be directlyinvolved in the formation of SAHF at the chromatin level and additionally be involved insenescence induction.

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Abstracts - Poster

Abstracts – Poster


Helena Ahlfors Abstract P2A novel player in T helper cell differentiation

Helena Ahlfors1, Soile Tuomela1,2, Tiina Henttinen1, Riikka Lund1 and RiittaLahesmaa1

1Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku,Finland, 2Turku Graduate School for Biomedical Sciences, Turku, Finland

Naïve CD4+ T helper lymphocytes can differentiate into two distinct subsets, termed as Th1and Th2, according their cytokine expression profiles. These subsets of T helper cells areresponsible for specific immune functions; Th1 cells contribute to cell-mediatedinflammatory immunity, while Th2 cells are responsible for humoral responses. Defects inthe T helper cell differentiation may result in the pathogenesis of various immune mediateddiseases such as asthma and allergies. We have carried out genome wide gene expressionprofiling during various stages of Th cell polarization to Th1 or Th2 cells to identify potentialnew players involved in the process. In this study we have focused on one Th2-specifictranscription factor (acronym TF2). The differential expression of TF2 observed withmicroarray experiments was confirmed both at mRNA and protein level during the early Th1and Th2 cell polarization. TF2 is clearly induced by interleukin-4 at early time points both onmRNA and protein level, and its expression remains at increased level throughout the earlydifferentiation process. As STAT6 plays a key role in Th2 cell differentiation, we investigatedwhether downregulation of STAT6 would regulate the expression of TF2. We transfectedprimary human Th cells with plasmid-based constructs of STAT6 siRNAs contaning amarker for cell selection and cultured the cells in polarizing conditions. Here we show thatdownregulation of STAT6 by siRNA downregulates the expression of TF2. Characterizationof putative STAT6 binding sites in the promoter region of TF2 is in progress. In addition, wedesigned and optimized vector-based siRNAs to downregulate the expression of TF2.These constructs were transfected to primary human Th cells to elucidate the dose-dependent effects of TF2 in T helper cell differentiation, growth, proliferation and survival.After 24 and 48 hours and 7 days of polarization the cytokine production of the transfectedcells was analyzed. Interestingly, the knockdown of TF2 changed the cytokine productionprofiles of cells polarized to Th1 and Th2 directions. Samples from the early time pointswere hybridized into Illumina beadarrays for genome wide detection of the genes regulatedby TF2. Studies in progress aim at further characterization of the role of TF2 in Th celldifferentiation.

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Barbara Alberter Abstract P3Histone modification pattern of the T lymphotropicHerpesvirus saimiri genome in latency

Barbara Alberter and Armin Ensser

Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany

Herpesvirus saimiri (HVS) is the prototypic γ2-herpesvirus. Its circular dsDNA genome (155kb) consists of an AT-rich coding region harbouring at least 77 open reading frames (orf)and a non-coding region which is made up of tandem repetitive elements with high GCcontent. After infection HVS establishes latency in its natural host, the new world primateSaimiri sciureus. Experimentally infected and thereby growth transformed human T cellsalso retain latent HVS genomes. Here, only the viral orf1 (bicistronic transcript, StpC andTip) and orf73 (LANA homolog) are transcribed. StpC and Tip are essential for thetransformation of T cells to antigen-independent growth and the orf73/LANA supports theepisomal maintenance of the viral genome in those cells. It is not known how geneexpression of latent genes on the one hand and repression of the major group of lyticgenes on the other hand are epigenetically regulated in γ2-herpesviruses. In this study, weperformed chromatin immunoprecipitation with seven different acetylation or methylationspecific antibodies followed by quantitative SYBR Green PCR to profile the histonemodifications in the herpesviral genome. Four studied promoters of lytic genes carryrepressive marks, whereas the orf73 promoter revealed a variable modification pattern. Asexpected, the promoter of orf1 was found to be wrapped up in permissive chromatin, but toour surprise the most permissive chromatin structure was revealed in the non-codingrepetitive elements at the ends of the genome.

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Marco Alvarez Abstract P4Histone variant macroH2A is an epigenetic factor involved inthe modulation of ribosomal gene expression duringseasonal adaptation of carp fish

Pinto R., Bouvet P. 1, Dimitrov S. 2, Molina A., Vera M.I. and Alvarez M.

Departamento de Ciencias Biologicas, Universidad Andres Bello and Millennium Institute forFundamental and Applied Biology, Santiago, Chile, 1Ecole Normale Superieure, Lyon,France, 2Institut Albert Bonniot, Grenoble, France

Cyprinus carpio acclimatization is a process mediated through molecular mechanisms thatcoordinate a homeostatic state in response to cyclic environmental changes. Consequently,molecular and cellular functions are reprogramd during seasonal adaptation of the fish. This“phenotypic plasticity” is the result of fine and coordinated regulation of gene expression.MacroH2A is a histone variant associated with epigenetic mechanisms of gene silencing.Previously, we have reported that high levels of macroH2A correlate with hypermethylation ofthe carp rDNA gene promoter during winter, concomitant with a lower transcription level ofribosomal genes. Altogether these observations seem to suggest that histone variantmacroH2A could be involved into the seasonal regulation mechanisms of the carp ribosomalbiogenesis.In the present work, by means of chromatin immunoprecipitation (CHIP), we demonstratedthat macroH2A is present in the rDNA cistron during the cold season. Furthermore, real-time PCR analyses of these experiments confirmed that macroH2A is mainly enriched inthe promoter region of the rDNA compared to summer season. Moreover, we tested thenuclease accessibility of carp rDNA promoter and here we show that the enzymeaccessibility decrease during winter suggesting a more compact state of the chromatin. Inconclusion, we postulate that an epigenetic mechanism like histone replacement by itsvariants, particularly the replacement of H2A by macroH2A, plays a central role in theregulation of ribosomal genes expression in carp fish.

FONDECYT 1040197; DI-UNAB 37-04

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Terra G. Arnason Abstract P5Rsp5 is required for nuclear shuttling of the Snf1 kinasecomplex in yeast

Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A. Harkness

Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada

Chromatin assembly in yeast is regulated by a complex molecular network governed in partby the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) and the SCF. Wehave shown that Rsp5p, localized exclusively to the plasma membrane and adjacent tovacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors, such asthe SCF. The APC then initiates replication-independent, but CAF-I-dependent, chromatinassembly. Here, we demonstrate another mechanism leading to Rsp5p-dependent APCactivity. Mutation to RSP5 leads to increased histone H3 phosphorylation and decreasedhistone H3 acetylation at elevated temperatures. We show that the histone H3 kinase,Snf1p, is required for the rsp5 phenotype. Interestingly, we previously demonstrated that theSnf1 kinase complex, which shuttles across the nuclear membrane, is required for APCactivity. Thus, we propose that Rsp5p is required for the transit of Snf1p across the nuclearmembrane. In support of this theory, we show that GFP-tagged Snf1p, Snf4p (activatorsubunit) and Gal83p (localizing subunit) all fail to localize to the nucleus upon carbon stressin rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p, failed to exit the nucleusin rsp5 mutants. We next asked whether Snf4p, which requires ubiquitination for stabilityand function, requires Rsp5p or any of the Rsp5p associated E2 enzymes. In ubiquitincoimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4p bound to ubiquitin, butnot GST alone. We observed that carbon stress induced an increase in ubiquitinated GST-Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4p ubc5p cells, GST-Snf4pwas again recovered, but we failed to observe induction of ubiquitinated GST-Snf4p uponcarbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5p interacting protein, Rod1p,and iii) Ubc7p, an E2 that physically interacts with Rsp5p, on Snf4p ubiquitination will bediscussed.

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Stuart P. Atkinson Abstract P6Epigenetic mechanisms of pluripotency and differentiation

Stuart P. Atkinson and Anna Golebiewska

Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle uponTyne, NE1 3BZ. U.K.

A great deal of recent research has concentrated on the epigenetic basis of pluripotency ofhuman and mouse embryonic stem cells (ESCs) and studies have also suggested that thedifferences between various cell types may be due to differences in global epigeneticprofiles. Understanding the epigenetic differences between pluripotent cell, such as humanESCs, and differentiated cell lines may allow further understanding of the role epigeneticsplays in development. Human embryonal carcinoma (EC) cells are first utilised as a model.The chromatin environment of promoters of genes involved in pluripotency, self renewal andearly stages of differentiation were studied in great detail in undifferentiated EC cells aswell as after ATRA-mediated differentiation. An active chromatin configuration, such as highlevels of H3K4 methylation and histone acetylation, was observed in EC cells at thepromoters of genes involved in maintenance of pluripotency and self-renewal (e.g. Sox2,Oct4, and Nanog). A similar pattern was observed at the promoters of genes involved inearly stages of differentiation (e.g. Gata2, Pax6) with additional H3K27 methylationsuggesting that such genes were primed for expression, but still kept in repressed state inpluripotent EC cells. During differentiation, such promoter configurations become moreactive linking this to expression of such genes in differentiated ECs, whereas morerepressed pattern was observed at promoters of genes involved in pluripotency and self-renewal (e.g. presence of H3K9me2). These studies and the growing knowledge of theepigenetic state of hES cells could potentially be used to devise reprogramming strategiesto induce ‘stem-like’ phenotypes in differentiated cells through the modulation of epigeneticmechanisms. In the future these studies may facilitate the production of isogenic tissues forregenerative therapy without the ethical and logistical problems associated with therapeuticcloning in humans.

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Joanne L. Attema Abstract P7Epigenetic features of hematopoietic stem cells using smallnumbers of highly purified primary cells

Joanne L. Attema1, Peter Papathanasiou1, E. Camilla Forsberg1, Jian Xu2,Stephen T. Smale2 and Irving L. Weissman1

1Institute of Stem Cell Biology and Regenerative Medicine, Departments of Pathology andDevelopmental Biology, Stanford University School of Medicine, Stanford, California, U.S.A;2Howard Hughes Medical Institute, Molecular Biology Institute and Department ofMicrobiology, Immunology, and Molecular Genetics, University of California, Los Angeles,California, U.S.A.

Hematopoietic stem cells (HSC) are endowed with the ability to produce all blood celllineages through their capacity to self-renew and differentiate to descendent progenitorswith restricted potential. Recent studies have shown that HSC express many differentlineage-affiliated genes at low levels, leading to the hypothesis that epigenetic marks mayexist at these loci for their critical expression during differentiation. We investigated this byexamining histone and DNA modifications at lineage-affiliated genes in prospectivelypurified hematopoietic stem and progenitor cells. Here, we describe a method that allows forthe analysis of DNA and associated histones from as few as 50,000 primary cells. Wefound that histone modifications and unmethylated CpG dinucleotides co-localize acrossdefined regulatory regions of key lineage-affiliated genes in HSC that were eithermaintained or lost in the committed progenitors consistent with their expression. These datasupport a model in which epigenetic histone modifications are present at lineage-affiliatedgenes in HSC and could serve as an epigenetic-based mechanism that underliesmultipotency.

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Kristin Baetz Abstract P8NuA4 is a cellular “Hub”: an integrative map of physical andgenetic interactions mediated by the NuA4 histoneacetyltransferase

Leslie Mitchell, Wendan Chen, Maria Gerdes, Jean-Philippe Lambert,Daniel Figeys and Kristin Baetz

Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology andImmunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

NuA4 is the only essential histone acetyltransferase in the budding yeast Saccharomycescerevisiae and has established roles in transcription, DNA repair and faithful chromosomesegregation. NuA4 acetylates histone H4, H2A and the histone variant Htz1, however theexact molecular mechanisms by which NuA4 mediates its cellular processes are poorlyunderstood. Nor is it known if NuA4 has additional cellular roles or acetylation targets. Tobetter understand the cellular functions of NuA4 we have exploited the biochemical andgenetic amenabilities of the seven non-essential subunits of NuA4 – Eaf1, Eaf3, Eaf5,Eaf6, Eaf7, Yaf9 and Yng2. Physical and genetic interactions centered on the non-essentialsubunits of NuA4 were mapped at high resolution using systematic proteomic and genomicmethods. Physical interactions were identified using large scale affinity purification of NuA4complex in all seven non-essential NuA4 mutant backgrounds to establish the contributionof each subunit to NuA4 complex integrity and their role in mediating interactions with non-NuA4 proteins. Genetic interactions for each of the non-essential NuA4 subunits wereuncovered using genome-wide synthetic genetic array technology. An extended network,consisting of more then 300 genetic and physical interactions, was found to connect NuA4to a wide range of cellular functions and has identified several novel cellular roles for NuA4,including golgi-vacuole protein transport. The NuA4 network is helping to define roles foreach of the non-essential subunits of NuA4 and we determined that there is a directcorrelation between a subunit’s contribution to NuA4 complex integrity and the extent ofgenetic interactions identified. For example unlike most of the non-essential subunits, Eaf1is crucial to NuA4 complex stability and displays greater than100 genetic interactionssuggesting that Eaf1 may be a scaffold protein for the NuA4 complex. Our extensivenetwork indicates that NuA4 is a “hub” of fundamental importance in the cell.

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Slobodan Barbaric Abstract P9Chromatin remodeling activities at the yeast PHO84 promoter

B. Silic1, T. Luckenbach2, S. Stuerzl2, P. Korber2 and S. Barbaric1

1Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University ofZagreb, 10000 Zagreb, Croatia and 2Adolf-Butenandt-Institut, Universitaet Muenchen,Schillerstr. 44, 80336 Muenchen, Germany

The yeast PHO84 promoter, which is coregulated with the well studied PHO5 and PHO8promoters in response to phosphate availability, is the strongest promoter of the PHO family,containing five binding sites for the specific activator Pho4. Under repressive conditions thereis a short hypersensitive region in the promoter containing two closely positioned Pho4binding sites, but upon induction the promoter chromatin structure is altered, so that at leastone nucleosome upstream and one downstream from the hypersensitive region areremodeled. Remodeling of chromatin structure leads to histone depletion from the promoterregion. The rate of histone eviction and consequently the rate of promoter activation arestrongly delayed in mutants deleted for either Snf2 or Gcn5. Nonetheless, after prolongedinduction full activation is achieved, but in the absence of Snf2 chromatin remodeling is onlypartial, resulting in displacement of the downstream but not of the upstream nucleosome.Therefore, eviction of these two nucleosomes requires different chromatin remodelingactivities.In contrast to Snf2, Gcn5 is not required for efficient remodeling of both nucleosomes uponfull induction, either in the presence or absence of Snf2. Similarly to Gcn5, the absence ofIno80 also causes delay in the promoter activation without affecting the final extent ofchromatin remodeling and the same is true for the histone chaperone Asf1. Taken together,Gcn5, Ino80, or Asf1 affects only the rate of remodeling at the PHO84 promoter, similar aswe previously found for the PHO5 promoter. However, with respect to the requirement for Snf2,the PHO84 promoter chromatin structure possesses hybrid characteristics compared to thetwo coregulated promoters: the PHO8 is essentially dependent on Snf2 and at the PHO5 onlythe rate of chromatin remodeling is reduced in the absence of Snf2.We have also examined a possible role of the histone variant H2A.Z in regulation of chromatinremodeling at PHO promoters. Interestingly, the absence of H2A.Z has only a slight effect onactivation of the PHO5 promoter, but causes a strong delay in the activation kinetics of thePHO84 promoter. This provides additional evidence that chromatin remodeling process at thetwo coregulated promoters involves different mechanisms.

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Vivian Bardwell Abstract P10Polycomb group and SCF ubiquitin ligases are found in anovel BCOR complex that is recruited to BCL6 targets

Micah Gearhart, Connie Corcoran, Joseph Wamstad and Vivian Bardwell

Department of Genetics, Cell Biology and Development and the Cancer Center, Universityof Minnesota, Minneapolis, MN 55455, U.S.A.

The corepressor BCOR potentiates transcriptional repression by the proto-oncoproteinBCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemiafusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocar-diodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) andSkp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, aPosterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A.BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets,indicating that the BCOR complex employs PcG proteins to expand the repertoire ofenzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can targetPcG proteins to DNA. In addition, the BCOR complex contains components of a secondubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmuno-precipitates isoforms of FBXL10 which contain a JmjC domain that recently has beendetermined to have histone H3K36 demethylase activity. The recruitment of two distinctclasses of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCORuses a unique combination of epigenetic modifications to direct gene silencing.

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Amrita Basu Abstract P11Computational prediction of histone and non-histone proteins

A. Basu1, Y. Zhao2, S. Hake1, K. Rose3, B. Ueberheide3, D. Hunt3, C.D.Allis1 and E. Segal4

1Laboratory of Chromatin Biology, The Rockefeller University, New York NY 10021,U.S.A.,2Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX75390,U.S.A., 3Department of Chemistry, University of Virginia, Charlottesville, VA 22904,U.S.A., 4Department of Computer Science and Applied Mathematics, The WeizmannInstitute of Science, Rehovoth 76100, Israel

Acetylation, a well-studied post-translational modification,.plays essential regulatory roles ina broad spectrum of biological processes, notably gene regulation. Although many studieshave been contributed on the molecular mechanism of acetylation dynamics, the intrinsicfeatures of substrate site specificity are still elusive and remain to be critically defined to apoint where predictions of unknown acetylation sites can be made with reasonableaccuracy. Since many enzymatic processes that modify histones have been identified andcharacterized, it is highly probable that these histone-modifying enzymes target non-histones in mammalian cells, as illustrated by the p300/CBP requirement for p53 and HIV1acetylation in human cells (1,2). Whether or not these enzymes target the substrate in aspecific manner and whether sequence composition surrounding the modified amino acidsplays an important role remains to be elucidated. In this work, using a sequence basedbioinformatics approach, we show that there is a bias towards specific amino acidssurrounding an acetylated lysine that may result from HAT recognition in histones andnuclear non-histone proteins. We hope that our approaches can be used as a predictivemethod in identifying acetylated substrates in the human proteome.

Refs:(1) Gu W and Roeder R.G, Activation of p53 sequence-specific DNA binding by acetylationof the p53 C-terminal domain. Cell 1997. 90: pp. 595–606.(2) Kiernan R.E. et al., HIV-1 tat transcriptional activity is regulated by acetylation. EMBO J,1999, 18: pp. 6106–6118.

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Mark T. Bedford Abstract P12Screening for the methylated proteome

Donghang Cheng and Mark T. Bedford

University of Texas M.D. Anderson Cancer Center, Department of CarcinogenesisPO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A.

The coactivator associated arginine methyltransferase, CARM1, is recruited by manydifferent transcription factors as a positive regulator. To understand the mechanism by whichCARM1 functions, we sought to isolate its substrates. We developed a small-pool screeningapproach for this purpose and identified CA150, SAP49, SmB and U1C as splicing factorsthat are specifically methylated by CARM1. We further showed that CA150, a molecule thatlinks transcription to splicing, interacts with the tudor domain of the spinal muscular atrophyprotein SMN, in a CARM1-dependent fashion. Experiments with an exogenous splicingreporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping inan enzyme-dependent manner. The identification of splicing factors that are methylated byCARM1, and protein-protein interactions that are regulated by CARM1, strongly implicatethis enzyme in the regulation of alternative splicing and points towards its involvement inspinal muscular atrophy pathogenesis.

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Sukesh R. Bhaumik Abstract P13Regulation of transcriptional activation by mRNA cap-bindingcomplex in vivo

Pratibha Bajwa, Abhijit Shukla, Nadia Stanojevic and Sukesh R. Bhaumik

Department of Biochemistry and Molecular Biology, Southern Illinois University School ofMedicine, Carbondale, IL-62901, U.S.A.

Eukaryotic gene regulation is largely controlled at the level of transcriptional activation bygene-specific activators which function by stimulating the assembly of general transcriptionfactors to form a preinitiation complex (PIC) at the promoters of the active genes.Interestingly, we show here that mRNA cap-binding complex (CBC) dramatically stimulatesformation of the PIC assembly and subsequently transcription in vivo. However, CBC isrecruited to the active gene following formation of the PIC assembly. These results revealthat PIC and CBC are intimately coupled via their reciprocal synergism, providing a novelregulatory pathway of transcriptional activation by CBC in a positive feedback mechanism.

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Marjorie Brand Abstract P14The Ash2L/MLL2 methyltransferase complex is important forß-globin transcription during erythroid differentiation

Patrick Lai1, Jeffrey A. Ranish2, Celina Demers1, Gaetan Juban3, FrancoisMorle3, Ruedi Aebersold2,4, F. Jeffrey Dilworth1, Mark Groudine5 andMarjorie Brand1

1Sprott Center for Stem Cell Research, Ottawa Health Research Institute, Ottawa, ON,Canada, 2Institute for Systems Biology, Seattle, WA, USA, 3Centre de Genetique Moleculaireet Cellulaire, Villeurbanne, France, 4Institute for Molecular Systems Biology, Zurich,Switzerland, 5Fred Hutchinson Cancer Research Center, Seattle, WA, U.S.A.

The mammalian ß-globin locus is comprised of five clustered ß-like globin genes, whosedevelopmental-specific expression is regulated by the distal locus control region (LCR),located dozens of kilobases upstream of the genes. Methylation of histone H3 at lysine 4(H3-K4) occurs at the ßMaj-globin gene during erythroid differentiation and is dependentupon the hematopoietic-specific transcription factor NF-E2-p45. However, the enzymeresponsible for H3-K4 methylation at the ß-globin locus is unknown.

Here, we set out to identify methyltransferase(s) interacting with NF-E2-p45 in erythroidcells using immunoprecipitation and mass spectrometry. Strikingly we found that NF-E2-p45 associates with 2 distinct histone H3 methyltransferases: G9a which modifies lysine 9,and MLL2 which is specific for lysine 4. Interestingly, in vitro these 2 methyltransferasesdisplay competing activities, which appear to be regulated by the acetylation status of theirtarget, suggesting a new element to the histone-code. Using chromatinimmunoprecipitation, we then showed that, during erythroid differentiation, the MLL2complex is recruited to the ß-globin LCR, 38kb upstream of the ßMaj-Globin gene.Interestingly, MLL2 (but not the Ash2L subunit of the MLL2 complex) is also recruitedacross the entire ß-globin locus from the LCR to the transcribed ß-Majglobin area,suggesting that the H3-K4 methyltransferase is transported across the ß-globin locus via aspreading mechanism. Finally, in contrast to the recent proposition that trimethylation of H3-K4 and H3-K9 co-exists within the active ßMaj-globin gene, we demonstrate that these 2modifications are mutually exclusive, trimethylated H3-K4 being instead strongly correlatedto acetylated H3-K9. In fact, we found that the ß-Majglobin transcribed area (methylated onH3-K4 and acetylated on H3-K9) is flanked by regions that are enriched in trimethyl-H3-K9,while being poorly trimethylated on H3-K9 itself. This is in agreement with earliersuggestions that one role of trimethyl-H3-K4 is to prevent spreading of the repressive H3-K9 trimethyl mark. Thus altogether our results provide new insights into the regulation oftranscription via recruitment and spreading of the H3-K4 methyltransferase MLL2 to specificgenes during development.

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Lauren Buro Abstract P15Histone methylation patterns at interferon-gamma induciblegene loci

Lauren Buro and Melissa Henriksen

Department of Biological Sciences, Fordham University, Bronx, NY 10458, U.S.A.

Here we examine the lysine methylation profile of histone H3 (H3) at several interferongamma (IFN-() induced, STAT1 dependent gene loci. STATs are a family of transcriptionfactors that, in response to a variety of extracellular ligands, are rapidly and transientlyrecruited from their latent state in the cytoplasm to the nucleus where they drivetranscription of target genes, affecting growth, differentiation, homeostasis and the immuneresponse. The accessibility of the activated STAT to its DNA binding site and thesucceeding gene expression are intimately tied to chromatin structure. Post-translationalmodifications of the residues in the amino terminal tails of the lysine residues of histonesdefine the functional state of the chromatin. Methylation of lysine residues of H3 isdescribed as either activating or repressive of gene expression, with the activity correlatedto the position of the lysine residue (K4 or K9), the level of methylation (mono, di ortrimethylation) and the location of the nucleosome itself (promoter or transcribed regions,euchromatin or heterochromatin). Chromatin immunoprecipitation followed by real-timePCR revealed K4 dimethylation (K4M2) and K4 trimethylation (K4M3) primarily in thecontrol regions of all the IFN-( inducible genes assayed, independent of transcriptionalactivity. That such methylation was not dynamic suggests a role for these modifications onlyin poising these genes for transcription. Further experiments to determine if K4M2 and/orK4M3 are permissive for gene induction are underway. H3 K9 trimethylation (K9M3), amodification reportedly associated with transcription elongation was not observed in thecoding regions of the IFN-gamma inducible genes.

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Jill S. Butler Abstract P16CXXC-finger Protein 1 regulates Dnmt1 protein expression

Jill S. Butler and David G. Skalnik

Indiana University School of Medicine, Herman B Wells Center for Pediatric Research1044 W. Walnut Street, R4 Room 324, Indianapolis, IN 46202, U.S.A.

Cytosine methylation and histone tail modifications are two epigenetic modifications thatinfluence gene expression. Elucidation of epigenetic regulation is becoming increasinglyimportant as deregulation of epigenetic processes is observed in many diseases, includingcancer. The CXXC1 gene encodes CXXC finger protein 1 (CFP1), a transcriptional activatorthat specifically binds unmethylated CpG dinucleotides. This DNA binding activity of CFP1makes it unique in that most CpG binding proteins bind methylated CpG dinucleotides andfacilitate heterochromatin formation. CFP1 has recently been identified as a component ofthe mammalian SET1 histone H3 lysine 4 methyltransferase complex (Lee and Skalnik, JBC(2005) 280:41725-31). Disruption of CXXC1 in mice results in an early embryonic lethalphenotype (Carlone and Skalnik, MCB (2001) 21:7601-6), and embryonic stem (ES) cellslacking CFP1 exhibit multiple epigenetic defects including altered histone modifications andreduced global cytosine methylation (Carlone, et al, MCB (2005) 21:4881-91). DNAmethyltransferase 1 (Dnmt1) is the major source of maintenance DNA methyltransferaseactivity in mammalian cells and is primarily responsible for copying cytosine methylationpatterns during DNA replication. Dnmt1 protein level and DNA methyltransferase activityare decreased by ~50% in CXXC1-/- ES cells and are rescued by stable expression ofmurine CFP1. Northern blot analysis along with real-time PCR experiments revealedDnmt1 transcript level is elevated ~ 40% in CXXC1 -/- ES cells. Additionally,immunoprecipitation experiments revealed an interaction between CFP1 and Dnmt1 in vivo.Regulation of Dnmt1 protein level by CFP1 is the first example of reduced Dnmt1 proteinwithout direct disruption of Dnmt1 gene function. The functional significance of this novelintersection of epigenetic regulatory proteins is currently under investigation.

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Jim Cakouros Abstract P17Identification of a novel enzyme which regulates the kineticsof histone arginine methylation in Drosophila melanogaster

D. Cakouros, T. Daish and S. Kumar

Hanson Institue, Department of Haematology, Frome Road, Adelaide, Australia

The sequential modifications of histones form the basis of the histone code whichtranslates into either gene activation or repression. The dynamic fluctuations of histonemethylation occurs in response to specific signals, regulated by the interplay of histonemethyltransferases and histone demethylases. Nuclear receptors recruit a cohort of histonemodifying enzymes in response to ligand binding and regulate proliferation, differentiationand programd cell death (PCD). In Drosophila, coactivators for the nuclear ecdysonereceptor (EcR/UsP) have not been extensively examined. We have identified a novelcofactor for the EcR which is normally involved in amino acid catabolism. This enzymecontains two enzymatic domains in its amino and carboxyl termini respectively, bindsEcR/UsP directly and potentiates ecdysone mediated transcription. We found that it caninhibit H3-R17 dimethylation mediated by the drosophila methyltransferase CARMER(DART4) by directly binding the amino tail of histone H3 specifically and this inhibition isdependant on the cofactors ketoglutarate and NADH. In drosophila cells this proteinregulates the kinetics of H3-R17 dimethylation and knockdown by RNAi can compromiseecdysone mediated transcription and PCD. The in vivo function of this enzyme is currentlybeing examined in flies.

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Raymond Camahort Abstract P18Genome-wide analysis of the budding yeast histone variantCse4 reveals occupancy at a single centromeric nucleosomeas well as additional non-centromeric locations

Raymond Camahort1,2, Bing Li1, Brian Fleharty1, Jerry L. Workman1, ChrisSeidel1, Jennifer L. Gerton1,2

1Stowers Institute for Medical Research, Kansas City, MO 64110, U.S.A., 2Department ofBiochemistry and Molecular Biology, 4011 Wahl Hall East, 3901 Rainbow Blvd., Universityof Kansas Medical Center, Kansas City, KS 66160, U.S.A.

Cse4 is the S.cerevisiae centromeric H3 histone variant, also known as CENP-A. Thishistone variant is incorporated into nucleosomes that are located at centromeres in buddingyeast and are required for proper kinetochore assembly and chromosome segregation.Centromeres in budding yeasts are defined by a specific 125 bp sequence that contains theelements CDEI, CDEII, and CDEIII. We demonstrate the localization of Cse4 to the sixteencentromeres of budding yeast chromosomes, as expected, and additionally the surprisingresult that Cse4 nucleosomes are located at other regions in the yeast genome, mainlyrepetitive regions including telomeres, rDNA, and Ty elements. To verify the localization ofCse4 to these regions, we fused Cse4 to a transcriptional activation domain anddemonstrate that this chimeric protein can activate transcription from the rDNA and Tyelements. Using high resolution quantitative PCR we demonstrate that there is a singleCse4-containing nucleosome at centromeres in vivo and this nucleosome is positioned overcentromere sequences. Finally, using purified components, we assemble canonical andCse4-containing nucleosomes in vitro and demonstrate that they can assemble with highefficiency on non-centromeric DNA. Our results demonstrate that Cse4 alone is notsufficient to nucleate a kinetochore. We suggest that Cse4, in addition to its critical role atthe centromere, may participate in repressing DNA metabolic processes such astranscription and recombination.

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Dylan Carney Abstract P19The RAG2 PHD finger links the histone code to V(D)Jrecombination

Dylan Carney, Alex Kuo, Tina Christakos and Or Gozani

Gilbert Biological Sciences, 371 Serra Mall, Stanford, CA 94306, U.S.A.

The PHD finger (Plant Homeodomain) module is a signature chromatin-associated domainthat is found throughout eukaryotic proteomes and is mutated in several human diseases.One such PHD-containing protein, the Recombination Activating Gene 2 (RAG2), isnecessary for the V(D)J recombination reaction that is responsible for generatingimmunoglobulin and T-Cell Receptor (TCR) diversity among lymphocytes. Mutations withinthe RAG2 PHD finger have been implicated in Omenns Syndrome, which is a result of aninability to effectively carry out V(D)J recombination. Preliminary work has shown that theRAG2 PHD domain binds specifically to histone H3 trimethylated at lysine 4 (H3K4me3).Furthermore, we have shown that the very same mutation (W453R) implicated in severalcases of Omenns Syndrome abrogates this binding. Our work suggests that the recognitionof H3K4me3 by the RAG2 PHD finger plays an important role in V(D)J recombination andthus proper immune system function.

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Beverly S. Chilton Abstract P20Analysis of RUSH/SMARCA3 isoforms and their interactionswith Egr-1 and c-Rel in the regulation of transcription

Aveline Hewetson and Beverly S. Chilton

Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center,3601 4th Street – MS6540, Lubbock, TX 79430, U.S.A.

RUSH, a SWI/SNF-related regulator of chromatin, was cloned from rabbit endometrium. Steroid-dependent alternative splicing yields two distinct isoforms (alpha and beta). Alpha has seven highlyconserved DNA-dependent ATPase domains, and beta has four. Although both isoforms are capable ofDNA binding, no studies have addressed their individual involvement in any biological process. RUSH ishighly conserved in eukaryotes and was independently identified as HIP116 (human) and P113 (mouse).These names were replaced by the gene name, SMARCA3. In addition to alternative splicing,progesterone-induced transcriptional activation is mediated by a bipartite progesterone receptor (PRE)half-site/overlapping Y-box combination (-38/-26) in the proximal promoter (-162/+90). Activation byprogesterone is achieved via the PRE half-site, and attenuated by NF-Y binding to the Y-box. Repressionis also achieved at two GC-rich sequences in the proximal promoter and a distal RUSH site (-616/-611).At each GC-rich site, specific binding of Sp3 was 15-17-fold more abundant than Sp1. The rate ofcomplex disassociation (off rate) for the distal A site (-131/-126) with the consensus Sp bindingsequence (GGGGCGGGG) was half that of the proximal B site (-62/-53) with a variant (GGGGCGGAG)sequence. MatInspector (Genomatix) analysis revealed the complexity of the stronger A site. A putativeEgr-1/Sp/MAZ/MZF1 site on the positive strand overlaps a putative c-Rel site on the negative strand(matrix similarity values ( 0.91). Supershift assays with nuclear extracts from progesterone-treatedanimals confirmed the binding of each candidate protein to the composite A site. In contrast, only Sp3binds the A site in nuclear extracts from estrous animals, and protein binding to the B site is negligible. Atthe RUSH site, isoform-specific binding was demonstrated with supershift assays. Exclusive binding ofalpha was confirmed with isoform-specific antibodies and nuclear extract from progesterone-treatedanimals. Exclusive binding of beta to the same site was confirmed with isoform-specific antibodies andnuclear extract from estrous animals. TransSignal TF-TF Interaction Arrays showed strong physicalassociations between alpha and DNA-bound Egr-1 and c-Rel. No physical interactions occurred betweenalpha and DNA-bound Sp1, MZF1 or NF-Y. Supershift assays confirmed alpha interacts physically withEgr-1 and c-Rel bound to DNA at the A site, and conversely Egr-1 and c-Rel interact with alpha bound toDNA at the distal RUSH site. ChIP assays confirmed alpha interacts physically with Egr-1 and c-Rel ateach site in the transcriptionally active promoter. RUSH/Egr-1 and RUSH/c-Rel interactions werevisualized by confocal microscopy. The proposed model for this interaction includes the fact that theauthentic RUSH site is separated from the composite A site by 500-bp of 5(-sequence with a series ofalternating pyrimidine-purine (CA) elements, which are anisotropically flexible and promote DNA bending.Thus progesterone acts via the PRE to induce transactivation. Fine-tuning the magnitude of thisresponse includes isoform-specific autorepression in which newly synthesized alpha binds DNA in asequence selective manner, and interacts physically with ligand-bound Egr-1 and c-Rel in the proximalpromoter. Conversely, when the gene is silent (estrous), beta replaces alpha, and the Egr-1 and c-Relpartners are unavailable for binding interactions. Alternative splicing is a powerful means of generatingmacromolecular complexity. Understanding the functional capabilities of alternatively spliced isoforms ofspecific genes that are transcription factors with intrinsic helicase activity will enhance our understandingof isoform-distinctive mechanisms of transcriptional regulation.

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Alexandra Chittka Abstract P21Signalling by a novel p75 neurotrophin receptor interactingprotein, SC1/PRDM4

Alexandra Chittka1*, Juan Carlos Arevalo2, Maria Rodriguez-Guzman1,Pilar Perez3, Moses V. Chao2 and Michael Sendtner1

1*MRC centre for developmental neurobiology, King’s College, Guy’s Campus, LondonBridge, London SE1 1UL, 1Institute for Clinical Neurobiology, University of Wuerzburg,97080 Wuerzburg, Germany, 2Molecular Neurobiology Program, Skirball Institute ofBiomolecular Medicine, New York University School of Medicine, New York, NY 10016,3Instituto de Microbiologia Bioquimica, CSIC/Departamento de Microbiologia y Genetica,Universidad de Salamanca, 37007 Salamanca, Spain

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member ofthe positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET)domain-containing zinc finger protein family, and it has been shown to be regulated byserum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution,and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine(BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 andanalyzed the function of its various domains. We show that SC1 acts as a transcriptionalrepressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requiresa trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in acomplex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires thepresence of its zinc finger domains and the PR domain. Additionally, these two domains areinvolved in the efficient block of BrdU incorporation by SC1. The zinc finger domains arealso necessary to direct SC1’s nuclear localization. Lastly, SC1 represses the promoter of apromitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated bySC1.Currently, we are searching for additional target genes of SC1/PRDM4 to address themechanistic role it plays during the development of the nervous system.

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Leslie Chu Abstract P22Inheritance of epigenetic chromatin states

Leslie Chu and Joachim Li

University of California San Francisco, 600 16th Street, Genetech Hall, Rm S376, SanFrancisco, CA 94158 U.S.A.

In eukaryotes, DNA is packaged into either euchromatin or heterochromatin. Euchromatinis classically defined as actively transcribed regions of DNA, while heterochromatin isdefined as transcriptionally silenced regions of DNA. This regional silencing requires theformation of a highly compact chromatin structure. Once established, the silent chromatinstructure is stably maintained throughout the cell cycle and inherited in each subsequentgeneration. The inheritance of silent chromatin is surprisingly stable, given the stressesplaced on chromatin through out the cell cycle. Previous work has shown that in each cellcycle, DNA replication disrupts nucleosomes, the fundamental unit of chromatin. Thissuggests that DNA replication also disrupts the silent chromatin structure. During each cellcycle, however, transcriptional silencing remains intact. These studies imply that if DNAreplication disrupts silent chromatin, then the restoration of silencing is tightly coupled toreplication. Our studies focus on understanding if DNA replication disrupts silencing, whatserves as the molecular memory for silent chromatin and identifying proteins required forthe inheritance of transcriptional silencing.

Using Saccharomyces cerevisiae, we show that Sir1, a heterochromatin establishmentprotein, and Asf1, a nucleosome deposition factor, are required for the inheritance ofHMLalpha transcriptional silencing. We also show that progression through S phase, in theabsence of Sir1 and Asf1, disrupts transcriptional silencing. This finding demonstrates thatan S phase event, possibly DNA replication, perturbs silencing. We used chromatinimmunoprecipitation to analyze the chromatin structure of HMLalpha. Interestingly, whensilencing is not inherited (lost with progression through one cell cycle), an intermediatechromatin structure is formed. This intermediate structure contains both euchromatin andheterochromatin features. These remaining heterochromatin features may serve as amolecular memory to restore the silent chromatin structure. These results suggest that Sphase disrupts transcriptional silencing, resulting in an intermediate chromatin structure.This intermediate chromatin structure may provide the molecular memory that directs Sir1and Asf1 restorative silencing activity, resulting in the faithful inheritance of silencing.

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Mair Churchill Abstract P23Structural basis for the histone chaperone activity of Asf1

Mair E.A. Churchill, Christine M. English, Melissa W. Adkins, Joshua J.Carson and Jessica K. Tyler

Department of Pharmacology, Department of Biochemistry and Molecular Genetics, U.Colorado Health Sciences Center, U.S.A.

Asf1 is a highly conserved chaperone of histones H3/H4 that assembles or disassembleschromatin during transcription, replication, and repair. The structure of the globular domainof Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 showshow Asf1 binds the H3/H4 heterodimer, enveloping the C-terminus of histone H3 andphysically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C-terminus ofhistone H4 that forms a mini-beta sheet with histone H2A in the nucleosome, undergoes amajor conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta-sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histonechaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a strand-capture mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly /assembly that may be used ubiquitously by histone chaperones.

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Jeffrey Craig Abstract P24What makes centromeres localise and cluster in interphasenuclei?

Irina Solovei1, Claudia Weierich1, Tatyana Karamysheva1, Paul Canham2,K.H. Andy Choo3, Thomas Cremer1 and Jeffrey M. Craig2

1Biozentrum (LMU), Grosshaderner Str. 2, Planegg-Martinsried, 82152 Germany,2Epigenetics Research Laboratory and 3Chromosome and Chromatin Research Laboratory,Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road,Parkville, Victoria 3052, Australia

Centromeres lie at the heart of chromosomes where they choreograph the multiple eventsof cell division. Centromeres are also a prime example of intergenerational epigeneticinheritance – their chromatin structure is preserved from one generation to the next. Theycontain large regions of heterochromatin and this has been shown to play a major role inestablishing structured nuclear domains which can control gene expression. In mammals,these domains are composed of clusters of centromeres and are often located close to thenuclear periphery. Furthermore, centromere are also localise to the periphery of theirindividual chromosome territories. The roles of centromeres per se and of heterochromatinin this nuclear localisation and clustering are unclear. To separate out these roles we havelooked at the nuclear localisation of human centromeres without tandemly-repetitive DNA.These neocentromeres contain only small domains of pericentric heterochromatin. We havelooked at the three dimensional position of neocentromeres in the nuclei of human cell linesusing in situ hybridisation and immunolofluorescence using antibodies to centromereproteins. We found that consistently, neocentromeres exhibited the same localisation asrepetitive centromeres within a territory and within the whole nucleus. However, ongoingfindings are showing that neocentromeres may not cluster with other centromeres in theinterphase nucleus. These results imply that large regions of heterochromatin exert moreinfluence on centromere clustering and the creation of large heterochromatic domains ininterphase and less influence on positioning with chromosomes and nuclei.

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Valerie J. Crusselle-Davis Abstract P25Regulation of beta-globin expression through the recruitmentof chromatin modifying enzymes by TFII-I and USF

Valerie J. Crusselle-Davis, Archana Anantharaman, Tihomir Dodev andJurg Bungert

Department of Biochemistry and Molecular Biology, Powell Gene Therapy Center, Center forMammalian Genetics, and Shands Cancer Center, University of Florida, Gainesville, FL32610 U.S.A.

The human beta-globin locus contains five functional genes which are arranged in the orderof their developmental expression. Gene proximal cis-regulatory DNA elements andinteracting proteins restrict expression of the genes to the embryonic, fetal, or adult stagesof erythropoiesis. In addition, the relative order of the genes with respect to the locuscontrol region also contributes to the temporal regulation of the genes. To more fullyunderstand adult beta-globin gene regulation, we examined the downstream promoter andfound that transcription factors TFII-I and USF interact with elements within this region inerythroid cells. TFII-I was found to act as a repressor of beta-globin expression while USFproteins were found to act as activators of beta-globin expression. It is becomingincreasingly clear that one role DNA binding proteins play is to recruit co-activators or co-repressors that modify histones or mobilize nucleosomes at regulatory sites. Therefore, tounderstand the mechanism behind the regulation of beta-globin expression by TFII-I andUSF we investigated the recruitment of chromatin modifying enzymes to the beta-globingene locus by these proteins in both an embryonic and adult environment. TFII-I was foundto interact with HDAC3 exclusively in embryonic environment. Suz12, a component of thePolycomb group complexes 2,3, and 4 which contains histone methylase activity, was alsofound to interact at the beta-globin promoter in an embryonic environment but not in anadult environment suggesting a role in repression. USF was found to interact withactivators in an adult environment. The role of USF in beta-globin expression was alsofurther investigated in transgenic mice which express a dominant-negative protein to USFexclusively in erythroid cells.

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Foteini Davrazou Abstract P26Molecular mechanism of histone H3K4me3 recognition bythe PHD finger of ING2

Pedro V. Peña1, Foteini Davrazou1, Xiaobing Shi2, Kay L. Walter2, VladislavV. Verkhusha3, Or Gozani2, Rui Zhao4 and Tatiana G. Kutateladze1

1Department of Pharmacology, University of Colorado Health Sciences Center, Aurora,Colorado 80045, U.S.A. 2Department of Biological Sciences, Stanford University, Stanford,California 94305, U.S.A. 3Department of Anatomy and Structural Biology, Albert EinsteinCollege of Medicine, Bronx, New York 10461, U.S.A. 4Department of Biochemistry andMolecular Genetics, University of Colorado Health Sciences Center, Aurora, Colorado80045, U.S.A.

The PHD (plant homeodomain) finger is found in many chromatin remodeling complexeshowever its function remains unknown. We found that a subset of PHD fingers targets tri-methylated H3 histone (H3K4me3) tail representing a novel family of protein-effectors thatrecognize this epigenetic mark1,2. We have determined the structure of the PHD finger ofING2 (inhibitor of growth) tumor suppressor in complex with a histone H3K4me3 peptideand characterized its specificity toward post-translationally modified histone tails. TheH3K4me3 peptide is bound in an extended conformation in a deep and extensive bindingsite consisting of elements that are conserved among other PHD fingers. Thetrimethylammonium group of Lys 4 is recognized by aromatic residues of the domain,whereas the intermolecular hydrogen-bonding and complementary surface interactions,involving five peptide residues, account for the PHD finger’s high specificity and affinity.Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairsthe ability of ING2 to induce apoptosis, suggesting a novel tumor suppressive mechanism.Strong binding of other human ING and yeast YNG PHD fingers indicates that therecognition of the H3K4me3 histone code is a general function of this protein family.

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Eullia de Nadal Abstract P27Control of gene expression by the yeast Hog1 MAPK

Eullia de Nadal, Meritxell Zapater, Glria Mas, Nuria Noriega, lex Vendrell,Sergi Regot and Francesc Posas

Cell Signaling Unit, Departament de Cincies Experimentals i de la Salut, UniversitatPompeu Fabra (UPF), E-08003 Barcelona, Spain

Mitogen-activated protein kinase (MAP) cascades are common signaling modules found inboth higher and lower eukaryotic cells. Budding yeast has several MAP kinase cascadesone of which contains a relative of the p38 family of stress activated MAP kinases. Thiskinase coordinates cellular responses to increases in external osmolarity by inducingdiverse osmo-adaptative response. Recent genome-wide transcriptional studies revealedthat a great number of genes are regulated by osmotic stress in a Hog1 dependent manner,suggesting a key role for the MAP kinase in stress-induced gene expression. However, thereis not a uniform mechanism by which stress-activated MAP kinase modulates geneexpression. It has been reported that MAPK can modify gene regulation by directphosphorylation of transcription factors, activators and repressors, such Smp1 and Sko1proteins. Apart from the role of Hog1 in the modification of transcription factors, this kinaseis associated specifically to chromatin in stress responsive promoters. Binding of the MAPKis critical for RNA Pol II and chromatin remodeling factors recruitment to osmostressresponsive promoters and for gene expression. These data suggest a new dimension togene regulation by signaling kinases and it prompted us to further study alternativemechanisms by which the MAP kinase Hog1 could regulate osmostress gene expression.

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Roger Deal Abstract P28Repression of flowering in Arabidopsis thaliana requireshistone H2A.Z deposition by a putative SWR1 complex

Roger B. Deal, Christopher N. Topp, Elizabeth C. McKinney and RichardB. Meagher

Department of Genetics (Deal, McKinney, Meagher) and Department of Plant Biology(Topp), University of Georgia, Athens, GA 30602, U.S.A.

In addition to the bulk histones that package the nascent genome during S phase,eukaryotes also encode variant histones that are deposited independently of DNAreplication and serve to functionally specialize particular chromatin regions. The histonevariant H2A.Z is universally conserved and has been implicated in a wide variety ofchromatin-mediated processes including transcriptional activation and euchromatinmaintenance in yeast, and heterochromatin formation in metazoans. In budding yeast andhumans H2A.Z is deposited into chromatin through the action of a conserved proteincomplex known as SWR1 or SRCAP, respectively. Here we show that the Arabidopsisthaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN 6(ARP6) and the Snf2 protein PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1(PIE1), are part of a single protein complex and are each required for deposition of H2A.Zinto chromatin at multiple loci. One of these loci is the FLOWERING LOCUS C (FLC) gene,a central repressor of the transition from vegetative to reproductive development. Loss ofH2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression andpremature flowering, indicating that H2A.Z is required for transcriptional activation of FLC tolevels that inhibit flowering. Collectively these results support the existence of a SWR1-likecomplex in Arabidopsis thaliana and show that, similar to its role in yeast, H2A.Z can serveto potentiate transcriptional activation in plants.

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Laurent Delva Abstract P29The Transcription Intermediary Factor 2 is required forzebrafish development

Julia Zhuravleva1, Bernard Thisse2, Christine Thisse2, Jean-Noël Bastie1

and Laurent Delva1

1Inserm U517, Faculté de Médecine, Université de Bourgogne, 7, bd Jeanne d’Arc, 21000Dijon, France, 2Institut de Génétique et de Biologie Moléculaire et Cellulaire,CNRS/Inserm/ULP, 1, rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France

The TIF2 (Transcription Intermediary Factor 2) gene, encoding a histone acetyl transferase,belongs to the p160 family. TIF2 interacts with liganded nuclear receptors, enhancingtranscription activity of the receptors. Because of partial genetic compensation effect, knockout TIF2 mice did not inform completely about the role of TIF2 in development. Therefore,we decided to use zebrafish as an animal model. tif2 is ubiquitously expressed in zebrafishdevelopment. tif2-knock down zebrafish embryos present embryonic alterations such as asmaller tail size, abnormalities in the notochord and mesenchyme. In addition, tif2morphants did not harbor posterior intermediate cell mass (ICM) indicating putativehematopoietic differentiation defects. To better understand the nature of the defectsobserved in tif2 morphants, we performed molecular analysis of various differentiationmarkers by using whole-mount RNA in situ hybridization. Our results suggest theinvolvement of tif2 in embryonic development, particularly in primitive hematopoiesis.Furthermore, we observed that loss-of-function of zebrafish tif2 results in massive apoptosisin the mesenchyme.

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Luisa Di Stefano Abstract P30Lsd1 mutation in Drosophila disrupt normal level of H3K4methylation and affects viability and fertility

Di Stefano L, Moon NS, Ji JY and Dyson N.

Massachusetts General Hospital Cancer Research Center and Harvard Medical School,Charlestown, Massachusetts, U.S.A.

Covalent modifications of histone tails have fundamental roles in determining the chromatinstructure and in regulating gene expression. One such modification, histone lysinemethylation was considered irreversible until the recent discovery of histone demethylases.Two distinct classes of histone lysine demethylases have been characterized so far, theJumonji-domain containing proteins and Lsd1. In mammalian cells, Lsd1 was shown tospecifically demethylate mono and di-methyl histone H3 lysine 4 and, when associated withthe androgen receptor, to act on dimethyl-H3K9. Lsd1 is highly conserved betweenorganisms from yeast to human but its role has yet to be studied in vivo. Here we describethe effects of Lsd1 mutation in Drosophila. We find that mutation of dLsd1 strongly affectsglobal level of methylation of mono and dimethyl H3K4 revealing that specificity towardsthese residues is conserved throughout evolution. In contrast the global levels of dimethyl-H3K9 are not affected in dLsd1 mutant suggesting either a functional difference to thehuman counterpart or that this regulation is restricted to specific tissues. As a consequenceof dLsd1 mutation, animal viability is strongly affected in a gender specific manner. dLsd1mutant flies are sterile and ovary development is strongly impaired. dLsd1 mutation stronglysuppresses positional effect variegation (PEV) and affects gene expression. Taken togetherour results support an important role for histone H3K4 lysine demethylation in the regulationof chromatin structure and gene transcription in Drosophila development.

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Stephan Diekmann Abstract P31In vivo dynamic (FRAP, FCS) and neighbourhood relation(AB-FRET, FLIM) studies of human inner kinetochoreproteins

S. Diekmann, P. Hemmerich, S. Orthaus, S. Weidtkamp-Peters andC. Hoischen

Molecular Biology, FLI, Beutenbergstr. 11, D-07745 Jena, Germany

The kinetochore specifies a DNA/protein assembly at the surface of chromosomes thatplays an essential role in faithful segregation of the genetic material. To gain a precisedynamic understanding of this complex, the mobility of GFP-tagged inner kinetochoreproteins CENP-A, CENP-B, CENP-C, CENP-H, CENP-I, and hMis12 were analyzed in livinghuman cells using fluorescence recovery after photobleaching (FRAP) and fluorescencecorrelation spectroscopy (FCS). In interphase cells, CENP-A, CENP-B, CENP-C, CENP-Hand CENP-I are stable components of the kinetochore over hours while hMis12 rapidly andcompletely exchanges within seconds. FCS detected soluble pools of kinetochore proteinsin interphase cells with protein-specific diffusion coefficients indicating the absence of pre-assembled kinetochore protein subcomplexes. During mitosis CENP-A, CENP-C, CENP-H,and CENP-I remain stably associated with the kinetochores, while CENP-B becomes mobileand, strikingly, hMis12 becomes completely immobilized at the kinetochores. Thus, unlike allother chromatin binding complexes analysed so far, the kinetochore is not maintained by aconstant flux of rapidly exchanging components but rather by a static assembly mechanism.Alterations in the mobility of specific kinetochore proteins such as Mis12, however, appearto be associated with the changing functional properties of kinetochores during mitosis.Many of the proteins involved in kinetochore formation are known, however, little informationis available on molecular structures and complex architecture although structuralphenomena seem to play an important role for kinetochore function. In addition to theirdynamic behaviour, we also determined the neighbourhood relation (in the < 10 nm range)of the inner kinetochore proteins CENP-A, CENP-B, CENP-C and CENP-I as well ashistones in living human HEp-2 cells by energy transfer (FRET and FLIM). The data can bewell explained by a centromeric chromatin 30 nm fiber model. Our results elucidate thearchitecture of the human inner kinetochore complex.

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Jeffrey Dilworth Abstract P32MEF2 helps establish muscle specific pattern of geneexpression by recruiting Trithorax Group proteins to specificpromoters

Shravanti Rampalli, Marjorie Brand and F. Jeffrey Dilworth

Sprott Stem Cell Research Center, Ottawa Health Research Institute, Ottawa, Ontario, K1H8L6., Canada

Pluripotency of stem cells is proposed to be due to the fact that all genes that have thepotential to be transcribed lie in open chromatin. As cells divide and differentiate, epigeneticmarking acts as a cellular memory to decide which genes should be expressed, orrepressed through chromatin condensation. These epigenetic signals are generated bypolycomb (repression) and trithorax (activation) group proteins which mark genes bymethylating hisone H3 on lysine residue 9/27 and 4 respectively. In an effort to understandhow the muscle specific pattern of gene expression is established during differentiation, weset out to determine whether trithorax proteins play a role in establishing muscle specificgene expression. Initially, we used chromatinimmunoprecipitation (ChIP) to show that upondifferentiation, several specific muscle specific genes are marked by H3K4 trimethylation. Todetermine how trithorax proteins are targeted to these promoters, we used co-immunoprecipitation and found that the Mef2 family of transcriptional regulators interact withthe trithorax group protein Ash2L. In addition, pull-down studies suggest that in cellularextracts from muscle cells, only specific Mef2 isoforms (Mef2C and Mef2D but not Mef2A)interact with Ash2L. Importantly, this interaction is significantly increased by pre-treatingMef2C, or Mef2D with p38 kinase in the presence of ATP, suggesting that phosphorylationof Mef2 proteins enhances its binding to Ash2L. Finally, we demonstrate by ChIP that thetiming of Histone H3K4 trimethylation (epigenetic mark established by Ash2L complex)during differentiation of muscle cells coincides with the binding of Mef2 to several musclespecific promoters. Thus, it appears that the transcriptional activator Mef2 is helpingestablish the muscle specific pattern of gene expression though epigenetic marking ofspecific genes.

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Ivana Djuretic Abstract P33T-bet and Runx3 cooperate to activate Interferon gammaand silence Interleukin-4 in T helper-1cells

Ivana Djuretic1, Ditsa Levanon2, Varda Negreanu2, Yoram Groner2, AnjanaRao1 and K. Mark Ansel1

1Harvard Medical School and the CBR Institute for Biomedical Research, Boston, MA, USA02115, U.S.A., 2The Weizmann Institute of Science, Rehovot, Israel 76100

Cell differentiation requires activation of lineage-appropriate genes and silencing of lineage-inappropriate genes. Naive CD4+ T cells can differentiate down one of the two effectorpathways: T helper-1 (Th1) or T helper-2 (Th2). T-bet, a T-box family transcription factor, is acentral regulator during Th1 differentiation because it directly activates Th1-specific genessuch as Interferon gamma (Ifng), and silences Th2-specific genes, such as Interleukin-4(Il4). Here we demonstrate that T-bet induces another transcription factor, Runx3, with whichit cooperates in both the activation of Ifng and silencing of Il4. Although each factor wascapable of functioning on their own, optimal gene activation and rtpression depended onthe presence of both T-bet and Runx3. The mechanim of repression likely involves a directco-operation on DNA, as the two factors were able to form a complex on the Il4 silencer-derived probe in vitro. Current studies are aimed in further defining the nature of T-bet/Runx3 cooperation, including their ability to cause context-dependent gene activationand repression. In addition, since Il4 silencing has been associated with the appearance ofH3K27 methylation, an important goal of this study will be to dettermine if directcooperation of T-bet and Runx3 mediates the process of H3K27-methylation dependentsilencing in the Il4 locus as well as other relevant loci.

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Tom Donndelinger Abstract P34Seeing cells in a new light: Improving resolution with ascientific approach to tissue processing

Tom Donndelinger and Elizabeth Oldenkamp

BI-Biomics, 1512 12th Ave Rd, Nampa, Idaho, U.S.A.

One of the greatest shortcomings of fluorescent microscopy is the lack of resolution.Fluorescence studies alone cannot be solid proof of sub-cellular localization. Although thisproblem will never be entirely overcome, our methods for tissue fixation combined withfluorescent technology return more conclusive results for localization studies with resolutionup to 2400x. We also show that superimposition of fluorescent images with H&E stainedimages creates a much more informative and cohesive picture of cell biology thantraditional images with DAPI counterstains. Further, our studies have revealedunprecedented nuclear and nucleolar detail as well as multiple novel cellular processes thathave never been seen through light microscopy. This technology, while a simple upgrade toan existing procedure, is revealing data that could seriously alter currently acceptedscientific paradigms.

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Bojan Drobic Abstract P35Characterization of Histone H3 kinases, MSK1 and MSK2

Bojan Drobic and James R. Davie

CancerCare MB, Manitoba Institute of Cell Biology, 675 McDermot Ave., Winnipeg,Manitoba, R3E 0V9, Canada

Stimulation of the Ras-MAPK signal transduction pathway by growth factors (EGF) orphorbol esters (TPA) in parental (10T1/2) and oncogene (H-ras)-transformed (Ciras-3)mouse fibroblasts induces rapid phosphorylation of histone H3. Phosphorylation of H3occurs on Ser10 and Ser28 in the NH2-terminal tail. This phosphorylation event isimplicated in the regulation of immediate early genes such as c-fos and c-jun. Constitutiveactivation of the Ras-MAPK pathway in ras-transformed mouse fibroblasts increasesphosphorylation of H3 at Ser10 and Ser28 and we have shown that this increase is due toenhanced activity of histone H3 kinase, mitogen- and stress-activated protein kinase 1(MSK1). Characterization of the MSK complex will be undertaken in 10T1/2, Ciras-3 andHEK293. Preliminary results demonstrate that MSK1 is associated with SWI/SNF ATPase(Brg1) and histone H3 acetyltransferase (HAT) [PCAF], as well as with c-Fos/c-Jun, p65sub-unit of NFKB, 14-3-3 proteins, but not with HDAC1. Furthermore, chromatin remodelingactivity of the MSK complex will be investigated by isolating the MSK complex from 10T1/2,Ciras-3 and HEK293 (cycling, serum-starved, TPA/EGF treated) cells and performing HATand mononucleosome disruption assays. Preliminary HAT assay results suggest that theMSK complex contains HAT activity. Dynamics of MSK1/2 association with c-fos and c-junpromoters will be investigated via ChIP assay. Since the Ras-MAPK signaling is frequentlyderegulated in cancer (30% of human cancers contain aberrant ras), characterization of theRas-MAPK activated MSK complex and its associating activities (chromatin remodeling andmodifying activities) could provide a basis for the assessment of MSK as a noveltherapeutic target to treat cancer.

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Danielle Ellis Abstract P36Histone acetylation of SRC and p21 promoters in responseto histone deacetylase inhibitor treatment; implications ofHDAC activity and SRC expression

Danielle Ellis1 and Keith Bonham2

1Department of Biochemistry, University of Saskatchewan. and 2Cancer Research Unit,Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon,SK, Canada, S7N 4H4

Histone deacetylase (HDAC) inhibitors (HDIs), such as trichostatin A (TSA), are welldocumented for their ability to induce apoptosis, growth arrest and differentiation in a varietyof neoplasms. These effects are mediated through the activation and/or repression of geneexpression. Some classic examples of this phenomenon include the transcriptionalactivation of p21WAF1 and the transcriptional repression of SRC. Currently, it is believedthat HDAC inhibitors act at the histone level to alter chromatin dynamics through theinactivation of HDACs thereby resulting in histone hyperacetylation and increasedtranscriptional activation. However, transcriptional repression of gene expression is not soeasily explained by this model. Indeed, changes in the acetylation status of histonesassociated with genes repressed by HDAC inhibitors, such as SRC, have not been reported.Therefore, we carried out a systematic investigation of the changes in histone H3 and H4acetylation status at the promoter regions of two genes differentially affected by HDACinhibitors. Treatment of HT29 colon cancer cells with TSA led to similar changes in theacetylation of discreet H3 and H4 lysine residues at the SRC1A, SRC1alpha and p21promoter regions. Differential promoter specific acetylation changes were also observed;whereby, the SRC1alpha and p21WAF1 promoter regions demonstrated differential changesin acetylation as compared to SRC1A. The observations that SRC is repressed by HDIs anddemonstrates rapid changes in histone acetylation upon HDI treatment suggests that anHDAC(s) may be localized at the SRC promoter regions and may be involved in SRCactivation. Through the use of RNA interference, we observed that the knockdown of eachclass I HDAC (HDAC 1, 2, 3 and 8) did not repress SRC but actually resulted in increasedSRC expression. Taken together these results suggest that histone acetylation is not asimple predictor of promoter activity and class I HDACs have an inhibitory affect on SRCexpression. Further study of class II HDACs may aid in elucidating the mechanism by whichSRC is repressed by HDIs.

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Alexander Erkine Abstract P37Differential mechanisms of nucleosome displacement atyeast heat shock gene promoters

T. Y. Erkina and A. M. Erkine

Division of Basic Biomedical Sciences, Sanford School of Medicine, University of SouthDakota, 414 E. Clark St., Vermillion, SD 57069-2390, U.S.A.

Chromatin remodeling at promoters of co-regulated and highly inducible heat shock genesHSP12, HSP82, and SSA4 is characterized by robust histone displacement during inductionof transcription. These promoters reach their maximally histone stripped state via twodistinct chromatin remodeling pathways: one associated (for the HSP12 and HSP82promoters) and the other one not associated (for the SSA4 promoter) with robust histoneH3 specific acetylation. This observation implies that a histone acetylated platformrecognized by bromodomain containing chromatin remodeling complexes is not uniformlyrequired for the robust histone displacement at gene promoters during induction oftranscription. SNF2 deletion causes elimination of histone displacement from the HSP12promoter but not from the HSP82 and SSA4 promoters. Out of three analyzed heat shockgenes only HSP12 is characterized by inducible binding of HSF to the promoter, whileHSP82 and SSA4 have HSF preloaded before heat shock. The SNF2 deletion preventsHSF binding to the HSP12 promoter. Knowing that HSF cannot bind to chromatinized DNAwe speculate that in wild type cells the SWI/SNF complex determines sliding or relocation ofnucleosomes along the HSP12 promoter allowing HSF to establish promoter binding, whilein the snf2 mutant strain nucleosomes lose their dynamic behavior and block HSF loading,thus eliminating histone displacement.

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Ragnhild Eskeland Abstract P38HP1 binding to chromatin methylated at H3K9 is enhancedby auxiliary factors

Ragnhild Eskeland, Anton Eberharter and Axel Imhof

Adolf-Butenandt Institut, University of Munich, Schillerstr. 44, 80336 Muenchen Germany

A large portion of the eukaryotic genome is packaged into transcriptionally silentheterochromatin. Several factors have been identified that play important roles during theestablishment and maintenance of this condensed form. Methylation of lysine 9 withinhistone H3 and the subsequent binding of the chromo-domain protein HP1 is thought toinitiate heterochromatin formation in vivo and to propagate a heterochromatic state throughseveral cell divisions. Here we analysed the binding of HP1 to methylated chromatin in afully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 onlyweakly binds to methylated chromatin. However, the addition of recombinant SU(VAR)proteins such as ACF1 or SU(VAR)3-9 facilitates HP1 binding to chromatin methylated atH3K9. We propose that HP1 has multiple target sites that contribute to its recognition ofchromatin only one of them being H3K9me. These findings have implications for themechanisms of how specific chromatin modifications are recognized in vivo.

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George Feehery Abstract P39CpG methylated DNA standards and control primers for usein methyl sensitive PCR and bisulphite sequencing

George R. Feehery, Pierre-Olivier Esteve, Hang Gyeong Chin andSriharsa Pradhan

New England Biolabs, 240 County Road, Ipswich, MA 01938-2723. U.S.A.

Methylation-specific PCR (MSP), is a technology for the sensitive detection of genemethylation in the vertebrate genome. The procedure employs an initial bisulfite reactionthat modifies the genomic DNA, or converts unmethylated cytosines to uracils while 5-methylcytosines remain unaltered. The bisulphate modified DNA is amplified by PCR withspecific primers designed to distinguish methylated from unmethylated sequences. Becausethis is a sensitive PCR-based assay, the use of DNA and primer controls are necessary todetermine the quality of the bisulfite conversion, the identification of artifacts such asprimer-dimer pairing, and mispriming to undesired target DNA that can cloud theinterpretation of results.To create a methylation-positive DNA control, we have enzymatically methylated Hela, NIH-3T3, and Jurkat genomic DNA with a prokaryotic CpG Methylase (M.SssI). All the cytosineresidues (C) within the double-stranded dinucleotide recognition sequence 5?CG?3 aremethylated cytosine (C5). To test the extent of CpG methylation, we challenged the variousmodified DNAs with [3H]AdoMet and an excess of bacterial methyltransferase M.SssI. Nofurther incorporation of tritiated AdoMet was observed in the DNA substrates even afterextensive overnight incubation. MSP-PCR using 10 different primer sets also revealedcomplete methylation of all CpG dinucleotides. Bisulphite sequencing of methylated DNAdisplayed ~99.5% of CpG methylation of candidate genes.We created a reduced methylation DNA control by incubating Jurkat cells with a 2?Mconcentration of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-Azadc) for eightdays. The genomic DNA derived from cells treated with this drug exhibited some lowermolecular weight smearing when visualized on a 0.8% agarose gel. Bisulfite conversion andsequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normallymethylated revealed significant CpG demethylation. Used in conjunction CpG methylatedand unmethylated genomic DNA controls may serve as powerful tools in the investigation ofDNA methylation in the genome.

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Barna Fodor Abstract P40Identification of novel pericentric proteins by their localization

Barna D. Fodor, Mario Richter, Manuela Scaranaro and Thomas Jenuwein

Research Institute of Molecular Pathology (IMP) Dr. Bohrgasse 7, A-1030 Vienna, Austria

The eukaryotic genome is organized into distinct euchromatic and heterochromaticsubdomains. Heterochromatin has fundamental roles in the structural organization ofchromosomes, genome stability, and controlling epigenetic programs. Genetic screens in S.pombe and Drosophila have identified a number of genes called suppressors of variegation[Su(var)s] most of which encode components of heterochromatin. These screens exploitthe transcriptional silencing effect of heterochromatin on juxtaposed reporter genes, whichis compromised in Su(var) mutants (Schotta et al. 2003). Another principle to discriminateheterochromatin components is their characteristic localization in cells. In mouse cellspericentric heterochromatin forms typical (DAPI dense) foci. Co-localization of epitopetagged factors to these foci can easily be detected. Localization screens based on thisprinciple, were also successful in identifying heterochromatin associated proteins (Dellaireet al. 2003). However, the potential of this approach was not yet fully exploited.In this study we applied genetrapping to identify novel components of heterochromatin. Thetransduced genetrap constructs randomly integrate in the genome, generating translationalfusions of endogenous loci and the GFP transgene. We observed characteristic localizationpatterns of the expressed GFP fusion product for a number of clones, proving the utility ofour experimental design. The interesting clones were picked, and expanded. The identity ofthe trapped genes can be determined by 5’- and 3’-races. We are now extending our studiesto a number of cell lines with a variety of genetrap constructs.

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Maria Fousteri Abstract P41Cockayne syndrome A and B proteins differentially regulaterecruitment of chromatin remodeling and repair factors tostalled RNA polymerase II in vivo

Anne Jensen and Leon H.F. Mullenders

Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333RC Leiden, The Netherlands

Restoration of UV-inhibited transcription requires removal of transcription-blocking DNAlesions by transcription-coupled repair (TCR), a specialized repair pathway that removes UVinduced photolesions from transcriptional active genes. In mammals TCR is dependent onCSA and CSB proteins, however, their function and interactions are not well understood.CSA is a WD-40 repeat protein and a component of an E3-ubiquitin ligase, while CSB is aDNA-dependent ATPase that shares homology with SW2/SNF2 chromatin remodelers.Mutations in any of the two genes that encode for CSB and CSA lead to a rare recessiveneuro-developmental progeroid-like disorder, Cockayne syndrome (CS).Currently, information is lacking on the exact composition and molecular interactions of anactive TCR complex. Our aim was to improve understanding of TCR in vivo by isolation andanalysis of lesion-stalled transcription elongation complexes. We have used in vivocrosslinking and ChIP and we examined the recruitment of specific repair and chromatinremodeling factors to a UV-stalled RNAPII in TCR-proficient and -deficient human cells. Theprotocol was modified in such a way that it enabled the analysis of co-immunoprecipitatedproteins that reside in close proximity on damage containing chromatin fragments. Ourstudy revealed that CSB and CSA display differential roles in recruitment of TCR-specificfactors and that assembly for TCR in vivo occurs without disruption of the UV-stalledRNAPIIo. CSB fulfills a key role as coupling factor in the assembly of a chromatin-boundTCR complex that involves histone acetyltransferase p300, nucleotide excision repair (NER)proteins and CSA-DDB1 E3-ubiquitin ligase complex with the COP9 signalosome. CSA isdispensable for attraction of NER proteins, yet is required to recruit XAB2, the nucleosomalbinding protein HMGN1 and transcription cleavage factor TFIIS. This approach highlightsthe essential roles of CS proteins in TCR complex formation and provides a molecular linkbetween damage recognition, chromatin remodelling and NER.

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Robert Gillespie Abstract P42Retinoid regulated association of transcriptional coregulatorsand the polycomb group protein SUZ12 with the retinoic acidresponse elements of Hoxa1, RARß2, and Cyp26A1 in F9embryonal carcinoma cells

Robert F. Gillespie and Lorraine J. Gudas

Molecular Biology Program of Weill Graduate School of Medical Sciences andPharmacology Department of Weill Medical College of Cornell University, New York, NewYork 10021, U.S.A.

Hox gene expression is activated by retinoic acid (RA) binding to Retinoic Acid Receptor-Retinoid X Receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) oftarget genes. Hox genes are also repressed by polycomb group proteins (PcG), though howthese proteins are targeted is unclear. We used chromatin immunoprecipitation assays toinvestigate the association of RXRα, cofactors, and the PcG protein SUZ12 with the Hoxa1,RARß2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells during RA treatment. Wedemonstrate that the association of RARγ-RXRα with RAREs before and during RAtreatment remains relatively constant. pCIP, p300, and RNA polymerase II levels at targetRAREs also varied by gene, though RA increased the associaton of these proteins withRAREs. Conversely, SUZ12 was associated with all RAREs studied and this association wasattenuated by RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2,and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalentdomain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreasedat target RAREs whereas H3ac and H3K4me2 levels remained constant. These studiesprovide insight into the dynamics of association of coregulators with RAREs anddemonstrate a novel link between RA signaling and PcG repression.

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Clara Goday Abstract P43Chromatin modifications in germline chromosomes ofsciarid flies

P.G. Greciano and C. Goday

Ramiro de Maeztu 9, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain

A classic example of programd chromosome elimination and genomic imprinting is foundin sciarid flies (Diptera, Sciaridae), where whole chromosomes of paternal origin areselectively discarded from the genome during development. In early germ cells a singlepaternal X chromosome is eliminated in embryos of both sexes and in male meiotic cellsthe whole paternal complement is discarded. In sciarids, differential acetylation of histonesH3 and H4 occurs between chromosomes of different parental origin, both in early germnuclei and in male meiotic cells. We here investigated histone methylation modificationsbetween chromosomes in germline cells of Sciara ocellaris. In early germ nuclei, maternalchromosomes show high levels of di- and trimethylated histone H3 at lysine 4, whereasthis histone modification is not detected in paternal chromosomes. In male meiosis, onlythe eliminated paternal chromosomes exhibit high levels of di- and trimethylated histonesH3 at lysine 4 and dimethylated H4 at lysine 20. In early germ nuclei, RNA polymerase IIassociates to maternally-derived chromosomes but lacks phosphorylation of the carboxy-terminal domain on serine 2. The results suggest that histone H3 methylation at lysine 4does not correlates with transcriptional activity in early Sciara germline nuclei. Our resultssupports that specific covalent chromatin modifications such as histoneacetylation/methylation are involved in the imprinted behaviour of germline chromosomesin Sciara.

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Aaron D. Goldberg Abstract P44HIRA-dependent incorporation of histone H3.3 marks activegenes in mouse embryonic stem cells

Aaron D. Goldberg1, Fyodor D. Urnov4, Ileana M. Cristea2, ChingwenYang3, Jeffrey Miller4, Carlos Ramos5, Marco Seandel5,6, Daylon James5,Sandra B. Hake1,7, Peter J. Scambler8, Brian T. Chait2, Philip D. Gregory4,Shahin Rafii5,6,9 and C. David Allis1

1Laboratory of Chromatin Biology, 2Laboratory of Mass Spectrometry and Gaseous IonChemistry, and 3Gene Targeting Resource Center, The Rockefeller University, 1230 YorkAvenue, New York, NY, 10021, U.S.A. 4Sangamo BioSciences, Inc. Pt. Richmond TechCenter 501, Canal Blvd, Suite A100 Richmond, California 94804, U.S.A. 5Department ofGenetic Medicine, 6Department of Medicine, Division of Hematology-Medical Oncology,Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021,U.S.A. 7Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximiliams-Universitaet,Schillerstr. 44, 80336 Muenchen, Germany. 8Molecular Medicine Unit, Institute of ChildHealth, London WC1N 1EH, U.K. 9Howard Hughes Medical Institute, Weill Medical Collegeof Cornell University, 1300 York Avenue, New York, New York 10021, U.S.A.

The histone H3 variant H3.3 has been shown to be associated with transcriptionally active genes inmultiple organisms, and to be retained in some loci after transcription has ceased. (Ahmad andHenikoff 2002; Schwartz and Ahmad 2005) It is tempting to speculate that H3.3 associates withtranscriptionally permissive chromatin in differentiating mammalian cells, and that this localizationhas functional importance in the establishment or maintenance of gene activation. However, it isdifficult to address these questions due to the lack of available tools or antibodies to distinguishH3.3 from the other H3 variants H3.2 or H3.1. Here we describe a rapid and efficient method thatuses engineered zinc finger protein nucleases and a short, selection-less, promoter-less targetingconstruct to introduce an enhanced yellow fluorescent protein (EYFP) sequence into the C-terminalcoding exon of the endogenous histone H3.3B gene in mouse embryonic stem (ES) cells. In workin progress, we have used these heterozygous H3.3B-EYFP tagged ES cells to show that H3.3 isexcluded from H3K9me3-rich pericentric heterochromatin on metaphase chromosomes, andpartially but incompletely co-localizes with regions of H3K4me3. Through chromatinimmunoprecipitation (ChIP) assays, we show that H3.3 is significantly enriched at the promoters,depleted at the transcriptional start sites, and enriched into the 3’ ends of pluripotency genes Oct4and Nanog in undifferentiated ES cells. As a control for the specificity of H3.3 localization, we havealso used our method to introduce point mutations in H3.3B, generating EYFP-tagged H3.2expressed from the endogenous histone H3.3B locus. Unlike H3.3, H3.2-EYFP is diffuselylocalized throughout metaphase chromosomes, and is not excluded from pericentricheterochromatin. Finally, we have used our method to visualize the subcellular localization of H3.3in ES cells homozygous for a knockout of the putative H3.3 chaperone HIRA, demonstrating thatH3.3 incorporation into metaphase chromosomes is HIRA-dependent. These studies confirm andextend studies carried out in other non-mammalian models and provide a “proof-of-principle” of anapproach permitting the tagging of key chromatin constituents for analyses in mammalian cells.

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Elizabeth Goneska Abstract P45Phosphorylation of the SQ H2A.X motif is required for propermeiosis and mitosis in Tetrahymena thermophila

Xiaoyuan Song*1,3, Elizabeth Goneska*1,2, Qinghu Ren*1,4, Sean D. Taverna2,C. David Allis2 and Martin A. Gorovsky1

1Department of Biology, University of Rochester, Rochester, NY, U.S.A., 2Laboratory ofChromatin Biology, The Rockefeller University, New York, NY, U.S.A., 3School of Medicine,UCSD, La Jolla, CA, U.S.A., 4The Institute for Genomic Research, Rockville, MD,U.S.A.,*These authors contributed equally to this work

Phosphorylation of the H2A.X C-terminal SQ motif is required for efficient DNA double-strand break (DSB) repair in diverse organisms. Here we show that H2A.X, one of the twomajor H2As in Tetrahymena, is phosphorylated at serine 134 in response to DSBs inducedby chemical agents and during prophase of meiosis I. Using strains containing a mutation(S134A) that abolishes this phosphorylation, we demonstrate that phosphorylation of theSQ motif is required for normal micronuclear meiosis and mitosis, and to a lesser extent, fornormal amitotic macronuclear division. H2A.X phosphorylation is also important forTetrahymena cells to recover from exogenous DNA damage, and its absence, while notlethal, leads to extensive accumulation of DSBs in both micro- and macronuclei.

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Susana Gonzalo Abstract P46Telomere epigenetic modifications: a control of telomerelength and a stop on recombination

Susana Gonzalo1,3, Isabel Jaco1, Roberta Benetti1, Gunnar Schotta2, PeterKlatt1, Thomas Jenuwein2 and María A. Blasco1

1Telomeres and Telomerase Group. Molecular Oncology Program. Spanish National CancerCentre (CNIO). Madrid E-28029, Spain., 2Research Institute of Molecular Pathology (IMP).The Vienna Biocenter. A-1030 Vienna, Austria, 3Department of Radiation Oncology.Washington University School of Medicine. St. Louis, MO 63108, U.S.A.

Telomeres protect chromosome ends from being recognized as double strand breaks. Alterationsin the binding of telomere proteins or erosion of telomeric DNA below a critical threshold lead totelomere instability, activation of the DNA damage response pathway, cell cycle arrest, andsenescence or apoptosis. To avoid growth inhibition, cells maintain telomere length either bytelomerase or alternative lengthening of telomeres mechanism (ALT). Recently, we and otherinvestigators have demonstrated that telomere metabolism can also be regulated epigeneticmodifications. Our studies identified a number of repressive chromatin-modifying activities thatparticipate in the assembly of telomeric chromatin in mouse cells. In particular, the abrogation ofhistone methyltransferases (HMTases) Suv39h1 and h2, results in defective trimethylation oftelomeric histone H3 at lysine 9 (H3K9me3), leading to telomere elongation. These observations,together with a report showing telomere erosion upon heterochromatin protein 1 (HP1) over-expression in human cells, was the first demonstration that epigenetic alterations of mammaliantelomeres can lead to telomere length deregulation. Furthermore, loss of DNA methyltransferasesDnmt1 or Dnmt3a/3b, resulting in DNA hypomethylation of subtelomeres, or loss ofRetinoblastoma (Rb) family function, affecting trimethylation of telomeric histone H4 at lysine 20(H4K20me3) and global DNA methylation, lead to telomere elongation. Most recently, we haveobserved decreased telomeric H4K20me3 and telomere length deregulation in cells deficient forSuv4-20h1 and h2 HMTases, suggesting that these enzymes are responsible for this chromatinmodification at telomeres. Based on the available data, we propose the hypothesis that theacquisition of a heterochromatic “condensed” structure at the telomere restricts the access oftelomere elongating machineries in order to ensure telomere homeostasis. The “opening” oftelomeric chromatin by loss of repressive chromatin marks or an excessive heterochromatinizationby up-regulation of these marks could facilitate or further restrict the accessibility of telomereelongating activities, leading to telomere elongation or erosion, respectively. In fact, we haverecently found an increased frequency of telomeric sister chromatid exchange (T-SCE) events incells lacking Dnmt1 or Dnmt3a/3b, and also in cells lacking HMTases Suv39h1/h2 or Suv4-20h1/h2, by the Chromosome Orientation FISH technique (CO-FISH). This technique is a read-outof recombination among sister telomeres, a hallmark of activation of ALT mechanism of telomereelongation. In summary, we conclude that modifications of telomeric chromatin serve to controltelomere length and impose a stop on recombination.

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Tanya Gustafson Abstract P47Epigenetic silencing of Singleminded-2 in breast cancer

Tanya Gustafson1, Keelan Anderson1, Mike Kladde2 and Weston Porter1

1Texas A&M College of Veterinary Medicine, Department of Integrative Biosciences, CollegeStation, TX 77843, 2Texas A&M University, Department of Biochemistry and Biophysics,College Station, TX 77843, U.S.A.

Epigenetic gene regulation has been identified as a crucial event in breast cancerdevelopment. Epigenetic mechanisms of regulation include covalent modifications ofhistones and DNA methylation. These events occur throughout all stages of breasttumorigenesis and are a more common alternative to deletions or mutations for inactivatingbreast tumor suppressor genes. The human gene Singleminded-2 (Sim2) is a member ofthe basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of transcription factors, whichincludes genes responsible for maintenance of circadian rhythms (Per), sensors of hypoxia(Hif1α) and environmental contaminants (AhR). We have recently shown that Sim2 hastumor suppressor activity in the breast. Sim2 is expressed highly in normal breast cell lines,but expression is lost in invasive breast cancer cells. The purpose of this work is toelucidate the epigenetic mechanisms of Sim2 silencing in breast cancer cells.To determine whether Sim2 was silenced through epigenetic mechanisms, highly invasivebreast cancer cells (MDA435) and normal breast epithelial cells (MCF10A) were treatedwith a demethylating agent and a histone deacetylase inhibitor. Sim2 expression wasincreased by treatment in MDA435 cells, but did not change in MCF10A cells. Bisulfitesequencing of the CpG islands in MCF10A, MCF7 (mildly invasive breast cancer cells) andMDA435 cells revealed that methylation of a large island in exon 1 correlates with Sim2expression levels. Chromatin immunoprecipitation demonstrated that the Sim2 promoter ishyperacetylated in MCF10A cells and heterochromatic in MDA435 cells. The noveltechnique, MAP-IT, has been used to simultaneously characterize DNA methylation andchromatin structure on individual molecules. Overexpression of DNA methyltransferaseshas led to methylation, decreased expression and decreased histone acetylation of theSim2 gene. The data support Sim2 silencing through epigenetic mechanisms during breastcancer progression.

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Soon-Ki Han Abstract P48Role of plant CBP/p300-like genes in the regulation offlowering time

Soon-Ki Han1,3, Ju-Dong Song2,3 , Yoo-Sun Noh2,3 and Bosl Noh1,3

1Environmental Biotechnology National Core Research Center, Gyeongsang NationalUniversity, Jinju 660-701, Korea. 2Department of Biological Sciences, Seoul NationalUniversity, Seoul 151-742, Korea. 3Global Research Laboratory for Flowering at SNU andUW, Seoul National University, Seoul 151-742, Korea

CREB-binding protein (CBP) and its homolog p300 possess histone acetyltransferase (HAT)activity and function as key transcriptional coactivators in the regulation of gene expressionthat controls differentiation and development in animals. However, the role of CBP/p300-likegenes in plants is not yet elucidated. Here, we show that Arabidopsis CBP/p300-like genespromote flowering through affecting the expression of a major floral repressor FOWERINGLOCUS C (FLC). Although animal CBP/p300 generally function as coactivators, ArabidopsisCBP/p300-like proteins are required for the negative regulation of FLC. This CBP/p300-mediated FLC repression might involve reversible protein acetylation independent of histonemodification within FLC chromatin.

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Christin Hanigan Abstract P49Identification of an HDAC2 mutation in colorectal cancer andits consequences

Christin L Hanigan, Manon van Engeland, James Eshleman andJames Herman

Cancer Biology Program, Department of Oncology, The Sidney Kimmel ComprehensiveCancer Center at Johns Hopkins, Baltimore, MD 21231, U.S.A., Department of Pathology,University Maastricht, 6200 MD Maastricht, The Netherlands

Histone deacetylases are an important component of chromatin remodeling machineryassociated with transcriptional repression. Specifically, these proteins have been shown toaid in silencing tumor suppressor genes. While these proteins regulate a number of genesand cellular processes, we are only beginning to understand what genes and processesregulate them. One recent study showed mutations in the Wnt signaling pathway can leadto an upregulation of a histone deacetylase, HDAC2. Microsatellite instability can lead toincreased mutation rates in genes that contain mono, di, and tri-nucleotide repeat tracts.We have found taht HDAC2 has a poly(A) tract in exon1, which in some MSI+ colon cancercells is mutated leading to a frameshift and premature stop codon. We have observed theapoptotic resistance fo pharmacologically inhibiting HDACs in cell lines lacking HDAC2. Wepropose that HDAC2’s regulation of APAF-1 may play a role in this resistance. We havefound HDAC2 on the APAF-1 promoter and see up-regulation of APAF-1 at the mRNA levelonly in cell lines that have functional HDAC2 and are responsive to HDAC inhibition. Wesuggest that SAHA treatment up-regulates APAF-1 as one mechanism of inducing celldeath. Cell lines that have lost HDAC2 already have compensated for high levels of APAF-1 and are resistant to HDAC inhibition induced cell death.

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Troy Harkness Abstract P50Rsp5 is required for nuclear shuttling of the Snf1 kinasecomplex in yeast

Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A.Harkness

Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada

Chromatin assembly in yeast is regulated by a complex molecular network governed atleast in part by the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) andthe SCF. We have shown that Rsp5p, localized exclusively to the plasma membrane andadjacent to vacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors,such as the SCF. The APC then initiates replication-independent, but CAF-I-dependent,chromatin assembly. Here, we demonstrate another mechanism leading to Rsp5p-dependent APC activity. Mutation to RSP5 leads to increased histone H3 phosphorylationand decreased histone H3 acetylation at elevated temperatures. We show that the histoneH3 kinase, Snf1p, is required for the rsp5 phenotype. Interestingly, we previouslydemonstrated that the Snf1 kinase complex, which shuttles across the nuclear membrane,is required for APC activity. Thus, we propose that Rsp5p is required for the transit of Snf1pacross the nuclear membrane. In support of this theory, we show that GFP-tagged Snf1p,Snf4p (activator subunit) and Gal83p (localizing subunit) all fail to localize to the nucleusupon carbon stress in rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p,failed to exit the nucleus in rsp5 mutants. We next asked whether Snf4p, which requiresubiquitination for stability and function, requires Rsp5p or any of the Rsp5p associated E2enzymes. In ubiquitin coimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4pbound to ubiquitin, but not GST alone. We observed that carbon stress induced an increasein ubiquitinated GST-Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4∆ ubc5∆cells, GST-Snf4p was again recovered, but we failed to observe induction of ubiquitinatedGST-Snf4p upon carbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5pinteracting protein, Rod1p, and iii) Ubc7p, an E2 that physically interacts with Rsp5p, onSnf4p ubiquitination will be discussed.

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Tiffany Hung Abstract P51ING4 recognition of histone H3 trimethylated at lysine 4

Tiffany Hung, Xiaobing Shi and Or Gozani

Stanford University, 371 Serra Mall, Stanford, CA 94305, U.S.A.

ING4 is a candidate tumor suppressor protein and member of the evolutionarily conservedING (Inhibitor of Growth) family of chromatin-regulatory proteins. ING4 is a native subunitof an HBO1 histone acetyltransferase (HAT) complex and is thought to link HAT activity andtumor suppression. Here we present in vitro and in vivo evidence that the PHD finger (planthomeodomain) module of ING4 specifically recognizes histone H3 trimethylated at lysine 4(H3K4me3). This modification is an epigenetic hallmark of active transcription, and we aretesting the model that recognition of H3K4me3 by the ING4 PHD finger is important forgene activation via an increase in HBO1-dependent acetylation at nearby nucleosomes.

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David Johnson Abstract P52E2F1 and GCN5 facilitate the recruitment of nucleotideexcision repair factors to sites of UV-induced DNA damage

Ruifeng Gou, David L. Mitchell, Thomas R. Berton and David G. Johnson

The University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis, SciencePark-Research Division, PO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A.

The E2F1 transcription factor regulates the expression of genes involved in cell cycleprogression, apoptosis, differentiation, and DNA repair. In addition, emerging data suggeststhat E2F1 has a transcription-independent function in response to at least some forms ofDNA damage. We find that E2F1 localizes to sites of DNA damage caused by ultraviolet(UV) radiation and that this requires the ATR kinase and phosphorylation of E2F1 at serine31. In the absence of E2F1 the recruitment of nucleotide excision repair (NER) factors, suchas XPC and XPA, to sites of UV damage is impaired. This correlates with a defect in DNArepair and an increased sensitivity to UV-induced apoptosis in cells lacking E2F1. The GCN5histone acetyltransferase also accumulates at sites of UV-induced DNA damage. Moreover,GCN5 associates with E2F1 in response to UV exposure and knocking down E2F1expression impairs co-localization of GCN5 with damaged DNA. Like E2F1, GCN5 is alsoimportant for the efficient recruitment of repair proteins to sub-nuclear regions containing UVdamage. These findings indicate that phosphorylation mediated by ATR converts E2F1 into aDNA repair factor that localizes to UV damaged DNA. E2F1 then promotes DNA repair byrecruiting GCN5, and in turn, the NER machinery to sites of damage.

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Paul Kalitsis Abstract P53Nucleosome spacing analysis of repeat DNA regions in themouse genome

Paul Kalitsis, Sheena Rigby and K.H. Andy Choo

Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne 3052, Australia

Mammalian genomes consist of highly repeated satellite DNAs that are found in gene-poorregions such as centromeres and telomeres. In the mouse genome the proximal telomere iswithin 11 kb of the centromeric minor satellite DNA. These chromosome structures haveseparate and unique roles in chromosome segregation and stability. To investigate therelationship between these two domains we have examined the primary chromatin structureby partially digesting mouse chromatin with micrococcal nuclease from a variety of cell linesand tissues. Each digested chromatin extract was run on an agarose gel and hybridisedwith a representative repeat probe from telomeric, centromeric and peri-centromericdomains. Furthermore, we examined the effects on nucleosomal spacing in cell lines thatwere treated with chemical agents that perturb chromatin structure and differentiate cells.

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Min-Jeong Kang Abstract P54Role of a RPD3/HDA1 family histone deacetylase in theregulation of phytochrome-mediated light respases inArabidopsis

Min-Jeong Kang1,3 , Soon-Ki Han2,3, Yoo-Sun Noh1,3 and Bosl Noh2,3

1Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea.2Environmental Biotechnology National Core Research Center, Gyeongsang NationalUniversity, Jinju 660-701, Korea. 3Global Research Laboratory for Flowering at SNU andUW, Seoul National University, Seoul 151-742, Korea

Posttranslational acetylation of histone N-terminal tail is well known to influence genetranscription by changing chromatin structures: it relaxes the association of histone proteinswith DNA, displacing nucleosomes from the promoters of genes. Lately, histone acetylationhas also been thought to provide surfaces which transcription activators or repressorsrecognize and bind to. Histone acetylation is reversibly regulated by histone deacetylases(HDACs) that are categorized into three major groups; the RPD3/HDA1 superfamily, theSIR2 family, and the HD2 family. Arabidopsis has 10 members of HDACs belonging to theRPD3/HDA1 superfamily. In order to address the biological roles of the RPD3/HDA1 familyHDACs in plants, we isolated the loss of function mutants of Arabidopsis HDACs by areverse genetics and have characterized their phenotypes. We found the mutation in one ofthe HDACs causes hypersensitivity to the light-mediated inhibition of hypocotyl elongation.Genetic analyses showed the mutation in the photoreceptor phyB is epistatic to the hdacmutation in the hypocotyl elongation. More data indicating the role of the HDAC in theregulation of light responses will be presented.

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Panagiota Karagianni Abstract P55ICBP90, a putative link between histone ubiquitination andcell cycle progression

Panagiota Karagianni, Jun Qin and Jiemin Wong

Department of Molecular and Cellular Biology, Baylor College of Medicine, One BaylorPlaza, Houston TX 77030, U.S.A.

ICBP90 was initially identified as a transcription factor that can regulate Topoisomerase IIαexpression. The primary sequence of ICBP90 contains a Ubiquitin like, a SET and RINGAssociated (SRA), a PHD and two RING domains. It was recently shown that ICBP90exhibits auto-ubiquitination activity. Since a murine protein with high sequence identity withICBP90, Np95, has been shown to bind chromatin through the SRA domain as well asubiquitinate core histones in vitro, we wanted to examine if ICBP90 can also function as aubiquitin ligase for histones. We have found that ICBP90 can bind to chromatin andubiquitinate histone H3 both in vitro as well as in transfected cells, in a RING domain-dependent fashion. In fact, ICBP90 can poly-ubiquitinate H3 in vitro, which provides, to ourknowledge, the first paradigm of histone H3 poly-ubiquitination. By using massspectrometry we have mapped Lysine 79 of histone H3 as the site of covalent ubiquitinlinkage. ICBP90 has been previously shown to be required for G1/S as well as G2/Mtransition. We are currently addressing the potential role of ICBP90-mediated histoneubiquitination in cell cycle progression.

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Emmanuel Kas Abstract P56Altering the structure and functional properties ofheterochromatin with satellite-specific minor-groove binders

Roxane Blattes, Guillaume Susbielle, Caroline Monod and Emmanuel Kas

LBME - UMR5099 CNRS/UPS, IBCG, 118 route de Narbonne, 31062 Toulouse cedex 9, France

Repeated AT-rich sequences constitute an enormous target for DNA minor-groove binderssuch as oligopyrrole polyamides and diamidines. These sequence arrays are highlyenriched in pericentric heterochromatin, but also in the genomes of certain pathogenicparasitic microorganisms, or portions thereof as in the kinetoplast DNA of trypanosomes.We have previously shown that targeting of these sequences is easily achieved in vivo andis extraordinarily specific. The D1 protein of Drosophila melanogaster is associated with the359-bp 1.688 g/cm3 satellite III (SAT III) repeats that are cleaved by topoisomerase II (topoII) in vivo. We show that synthetic minor-groove binders that selectively target SAT IIIsequences alter the nuclear localization of topo II or interfere with its enzymatic activity toinduce modifications of position-effect variegation (PEV). P9, a satellite-specific polyamide,affects a D1-dependent pathway that directs topo II to the SAT III array and also perturbsthe association of HP1 with heterochromatin. In contrast, synthetic diamidines uncouple thetopo II/D1 interaction and cause a massive D1-independent relocalization of topoisomeraseII to AT-rich heterochromatin. This mobilization in turn results in a suppression of PEV. Wepropose that synthetic and natural AT-specific minor groove binders act coordinately withtopo II to effect assembly of specialized nucleoprotein structures such as heterochromatin.The antiparasitic diamidines used here also target AT-rich DNA sequences in their targetmicroorganisms. Their effects on the localization of topo II provide keys to understandingtheir mechanism of biological action, which most likely reflects an anti-genome activity thatinterferes with essential nucleoprotein complexes involving AT-rich sequences.

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Chul Geun Kim Abstract P57PIAS1 confers erythroid cell specific α-globin gene regulationby the CP2 transcription factor family

Ho Chul Kang, Kyung Sook Choi, Hyen Seok Heo, Tae Ho Jeon andChul Geun Kim

Department of Life Science, Hanyang University, Seoul 133-791, Korea

CP2c, a member of CP2 transcription factor family genes, was discovered initially in mouseas a transcription factor that binds to and stimulates transcription from the α-globinpromoter. We previously demonstrated that ubiquitously expressed CP2c exerts potenterythroid-specific transactivation of α-globin through interactions with CP2b, which isidentical to CP2a except that it has an additional 36 amino acids encoded by an extra exon,and protein inhibitor of activated STAT1 (PIAS1) (Kang et al., 2005). Indeed, significantreduction of α-globin expression was observed in RNAi-mediated knockdown of CP2c,CP2b or PIAS1 in erythroid cells, indicating that these three factors are indispensablecomponents for α-globin expression. However, the mechanisms by which how these threefactors confer erythroid-specific activation of α-globin in vivo are unsolved. Here we reportthat PIAS1 confers erythroid cell specific α-globin gene regulation by the CP2 transcriptionfactor family. We find that CP2c is mostly present in the cytoplasm, whereas both CP2b andPIAS1 are solely in the nucleus and CP2a is in the cytoplasm. Interestingly, overexpressionof CP2b or PIAS1 induces nuclear translocation of CP2c. Furthermore, the strong DNAbinding activity of CP2c which was destabilized by CP2b is restored by supplementation ofPIAS1 into the EMSA reaction, suggesting that PIAS1 induces the DNA/CP2b/CP2c/PIAS1quaternary complex formation in the nucleus. We confirmed that all three factors interactwith each other using their two discrete binding domains, and proteins containing minimallytwo tethered CP2b/CP2c binding domains of PIAS1 are sufficient to maintain the high levelof DNA binding and transcriptional activities of CP2b/CP2c. Taken together, our datasuggest that PIAS1 commands the protein-protein interaction, subcellular localization, andDNA binding abilities of the CP2 transcription factor family to confer erythroid cell specificα-globin gene regulation.

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Keun Il Kim Abstract P58A novel link between SUMO modification of a chromatinremodeling complex and cancer metastasis

Sung Hee Baek1, Jung Hwa Kim1, Hee June Choi1, Bogyou Kim1, Ji MinLee1, Ik Soo Kim1 and Keun Il Kim2

1Department of Biological Sciences, Seoul National University, Seoul 151-746, SouthKorea, 2Department of Biological Sciences, Sookmyung Womens University, Seoul 140-742, South Korea

Defining the functional modules with transcriptional regulatory factors that govern switchingbetween repression and activation events is a central issue in biology. We have reportedthe dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate ametastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumormetastasis, and further which signaling factors confer repressive function on reptin andhence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., NatureCell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealedthe presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptinthat underlies its repressor function. DeSUMOylation of reptin alters the repressive functionof reptin and its association with HDAC1. Further, SUMOylation status of reptin modulatesthe invasive activity in cancer cells with metastatic potential. This provides a clear definitionof the functional model and a novel insight for linking SUMO modification to cancermetastasis. As a follow-up study, we will address novel findings on the function of newlyidentified histone methyltransferase as a component of reptin, linking chromatin remodelingprocess and cancer metastasis.

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Sarah Kimmins Abstract P59Methylation of Histone H3 at lysine 4 is dynamic and tightlyregulated during male germ cell development

Véronik Auger, Maren Godmann and Sarah Kimmins

Departments of Animal Science and Pharmacology and Therapurtics, McGill University,Montreal, Canada

Idiopathic male infertility is associated with genetic and epigenetic abnormalities. Histonescan undergo epigenetic modifications on the N-terminus including methylation, acetylationand phosphorylation, among others. These histone modifications are hypothesized to signalchanges in chromatin structure leading to altered gene expression and recruitment ofregulatory transcription complexes. To date there is little information on the distribution andsignificance of histone modifications in spermatogenesis. We have identified the cellularlocalization patterns and developmental regulation of histone H3 mono-, di-, and tri-methylation at lysine 4 (K4), and the epigenetic modifiers implicated in the, removal andreading of these epigenetic marks namely, LSD1, MBD2a/b, and HDAC1, duringmammalian spermatogenesis. Testis were collected from the first wave of spermatogenesisin mice at postnatal days 6 (type A spermatogonia), 8 (type A and B spermatogonia), 10(pre-leptotene and leptotene), 12, (zygotene), 14 (early pachytene), and 20 (late pachytene)for immunolocalization and Western blot analysis. Patterns of distribution were furtherconfirmed using highly specific cell isolation and staging methods. Histone H3-K4 mono-,di- and tri-methylation is dynamic and widely distributed in spermatogenic cell types and isassociated with euchromatic regions. This strongly suggests that as in other tissues, in thetestis, methylation of histone H3 at lysine 4 serves key functions in the regulation of genetranscription. Expression of LSD1 is tightly regulated during germ cell differentiation.Remarkably, in comparison to somatic tissues, LSD1 is preferentially expressed in the testis.Interaction studies reveal unique transcriptional regulatory complexes associated with H3-K4 methylation in the testis including the association of LSD1 and MBD2b in a complex withHDAC1, presumably forming transcriptional repressor complexes. These studies serve toenhance our understanding of epigenetic control of the transcriptional program governingmale germ cell differentiation in normal and pathological states.

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Robert Klose Abstract P60JmjC-domain-containing proteins and histone demethylation

Robert J. Klose and Yi Zhang

Howard Hughes Medical Institute, Lineberger Comprehensive Cancer Center, Department ofBiochemistry and Biophysics, University of North Carolina at Chapel Hill, U.S.A.

Histone methylation plays important roles in regulation of gene expression, maintenance ofgenome integrity, and epigenetic inheritance. A wealth of understanding is available aboutthe histone methyltransferase enzymes which place these covalent modifications, but, untilrecently, enzymes capable of reversing histone methylation remained elusive and histonemethylation was thought to be a relatively static modification. The identification of lysinespecific demethylase1 (LSD1) revealed that histone methylation could be dynamicallyregulated in a manner similar to histone acetylation and phosphorylation. Recently theJumonji C (JmjC) domain has been shown to possesses Fe(II)/alpha-ketoglutaratedependent histone demethylase activity. Bioinformatic analysis of the extended JmjC-domain containing family of proteins has enabled us to categorize these proteins into sevenevolutionarily conserved groupings based on homology within the JmjC-domain and overallprotein domain architecture. By analysing the predicted co-factor binding sites withinindividual JmjC-domain groupings we have been able to utilize a targeted approach tocharacterize additional JmjC-domain containing histone demethylases. Based on ouranalysis we recently identified a novel histone demethylase, JHDM3A, which has thecapacity to reverse the tri-methyl lysine modification mark on histone H3K9/36. Ourcontinued functional analysis has revealed additional histone demethylases within the JmjC-domain containing family of proteins.

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Christoph M. Koch Abstract P61The landscape of activating histone modifications across 1%of the human genome

Christoph M. Koch, Robert M. Andrews, Paul Flicek, Shane C. Dillon,Ulas Karaoz, Gayle K. Clelland, Sarah Wilcox, Dave M Beare, Joanna C.Fowler, Phillippe Couttet, Keith D. James, Gregory C. Lefebvre, AlexanderW. Bruce, Oliver M. Dovey, Peter D. Ellis, Pawandeep Dhami, Cordelia F.Langford, Cordelia F. Langford, Zhiping Weng, Ewan Birney, Nigel P.Carter, David Vetrie and Ian Dunham

The Wellcome Trust Sanger Institute and European Bioinformatics Institute Wellcome TrustGenome Campus, Hinxton, Cambridge, U.K., Bioinformatics Program and BiomedicalEngineering Department Boston University, 24/44 Cummington St., Boston, MA 02215, U.S.A.

The NHGRI has established a pilot project (ENCODE) to explore computational andexperimental methods to develop an encyclopaedia of DNA elements in the humangenome. Initially the project targets 1% of the genome chosen according to the criteriaoutlined at http://www.genome.gov/10506161. We have constructed a microarrayrepresenting the 44 ENCODE regions consisting of 24005 PCR fragments with an averagesize of ~1 kb. It covers ~80% of the targeted regions including repetitive elements wherepossible. We are using this microarray to assay DNA samples enriched for sequencesinvolved in specific biological processes and functions generated by chromatinimmunoprecipitation (ChIP). ChIP experiments are being performed with a variety ofantibodies for specific histone modifications in a lymphoblastoid cell line (GM06990), anerythroleukemia cell line (K562) , foetal lung fibroblastoid cell lines (IMR90, HFL-1), cervixcarcinoma cell line (HeLaS3), a T-cell line (MOLT4) and a chimpanzee cell line PTR8. Wecorrelate maps of histone modifications with a range of genomic DNA features includingC+G content, genes/exons, repeat elements, SNP density and regions of conserved DNAsequence identified by comparative sequencing across multiple species as well as theexpression profiles of the cell lines. Preliminary analysis reveals strong enrichments ofacetylated histone H3 and di- and tri-methylated histone H3 (H3K4me2 and H3K4me3) at 5ends of transcriptional start sites. Mono-methylated histone H3 (H3K4me1) was found to bewidely distributed and not exclusive focussed to transcriptional start sites similar toacetylated histone H4 (H4ac). While comparing enrichments between different cell lines wefound a correlation of the expression status of genes and the absence or presence ofH3K4me3 at the transcriptional start site/promoter. Active promoters in each cell line show arobust enrichment of H3K4me3 while inactive promoter do not.

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Ryoki Kujiki Abstract P621alpha,25(OH)2D3-induced transrepression on 1alpha-hydroxylase gene promoter mediates chromatin remodelingthrough WINAC

R. Fujiki1, M. Kim1, Y. Sasaki1, H. Kitagawa1 and S. Kato1,2

1Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, Universityof Tokyo, Tokyo, Japan, 2ERATO, Japan Science and Technology Agency

Vitamin D receptor (VDR) is a nuclear receptor (NR) regulating bone metabolism andcalcium homeostasis. VDR modulates many target genes in a ligand-dependent manner.VDR-mediated gene expression requires a large number of co-regulator complexes.Recently, we identified WINAC as a novel VDR co-regulater complex that reconfiguresnucleosomal array around vitamin D response element (VDRE) (Cell, 113, 905, 2003).While mechanism of transactivation by NRs is well understood, the molecular basis of theligand-induced repression remains to be uncovered.To address this issue, we focused on the 1a,25(OH)2D3-induced transrepressionmechanism of 25(OH) D3 1a-hydroxylase [1a(OH)ase] gene, encoding a key enzyme ofvitamin D metabolism. The transactivation function by VDIR, a transcriptional factorrecognizing negative VDRE in 1a(OH)ase promoter (1anVDRE) (EMBO J. 23, 1598, 2004),was found to be suppressed by liganded VDR and/or WSTF, a WINAC major component. Inco-immunoprecipitation assay, WSTF physically interacts with both unliganded VDR andacetylated histones. ChIP assay showed that this interaction presumably allowed DNA-unbound VDR to associate with the 1anVDRE region prior to ligand binding. Using mouseembryonic fibloblasts from VDR knockout mice, we further found that VDR is required forthe association of WSTF with 1a(OH)ase promoter, implying 1anVDRE-specific associationof WSTF is defined by VDR. Next, we showed in vitro that the association of WSTF withacetylated histones is required for assembly of liganded VDR with VDIR bound to the1anVDRE. WSTF bromodomain was then mapped as interaction surface to acetylatedhistone H3. Interestingly, WSTF with deleted bromodomain acts as a dominant negativemutant in the transrepression of 1a(OH)ase gene. All together, WSTF association with bothunliganded VDR and acetylated nucleosomes, appears to be indispensable for this ligand-induced transrepression (EMBO J. 24, 3881, 2005). Thus, we have identified a novelmechanism of ligand-induced tranrepression by NRs that links transrepression andpromoter histone acetylation.

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Sharmistha Kundu Abstract P63SWI/SNF establishes transcriptional memory at theSaccharomyces cerevisiae GAL1 gene

Sharmistha Kundu, Peter J. Horn and Craig L. Peterson

Program in Molecular Medicine, University of Massachusetts Medical School, 373Plantation St., Biotech 2, Suite 210, Worcester, 01605, U.S.A.

Chromatin remodeling enzymes that chemically modify histones or DNA have long beenlinked with transcriptional memory in diverse eukaryotes. We show that the ATP-dependentchromatin remodeling enzyme, SWI/SNF is essential for establishing transcriptional memoryin the yeast, Saccharomyces cerevisiae. We observe memory in the regulation of the yeastGAL1 gene expression. Though SWI/SNF is dispensable for inducing GAL1 transcription, itsrecruitment allows much rapid kinetics of GAL1 reinduction, after being transitorilyrepressed with glucose. This phenomenon requires the ATPase activity of SWI/SNF and isepigenetically inherited by daughter cells. Significantly, SWI/SNF appears to antagonize theISWI complexes to establish transcriptional memory at GAL1. Upon further examination wefind that deleting the ISW2 complex specifically rescues growth defect of swi2- cells ingalactose and also rescues expression of another SWI/SNF dependent gene. Furtherexamples of SWI/SNF - ISW2 antagonism are presented.

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Georg Kustatscher Abstract P64Metabolite-sensitive and metabolite-insensitive chromatinsurfaces through the human histone macroH2A

Georg Kustatscher, Judith Sporn, Michael Hothorn, Bjoern Fritz, MiriamBortfeld, Klaus Scheffzek and Andreas Ladurner

Gene Expression and Structural Biology Unit, EMBL Heidelberg, Germany

Small-molecule metabolites play an important role in the regulation of gene expression. Wediscovered that human chromatin might be a receptor for NAD-metabolites produced by Sir2deacetylases. We are now studying whether this histone might provide a regulatoryconnection between metabolism and vertebrate chromatin.Unlike other deacetylases, Sir2 and its metazoan homologues use NAD to carry out thechemically straightforward deacetylation reaction. Sir2 may thus respond to the redox stateof a cell. In fact, evidence links Sir2 to metabolism, chromatin and aging by mediating thebenefits of life-extension through caloric restriction in yeast, C. elegans and Drosophila. Themammalian orthologue, SirT1, is induced under caloric restriction and regulates fat storageand the insulin pathway through PPARgamma and FOXO deacetylation. Sir2/SirT1 mightthus play a conserved role in the metabolic control of gene expression and inheterochromatin formation.We find that its metabolite, O-acetyl-ADP-ribose (AAR), binds the macro domain of thehuman histone macroH2A1.1. The novel crystal structure of the protein-nucleotide complexreveals how chromatin may be a direct target for endogenous metabolites. Further,macroH2A1 is subject to alternative splicing, where two mutually exclusive exons producetwo distinct proteins. Crucially, these exons encode a region critical for ligand-binding. Infact, the splice variant cannot bind AAR. Structural plasticity between the isoforms thusresults in proteins that adopt the same fold, but show binary sensitivity to AAR. Humanchromatin may thus bear metabolite-sensitive and metabolite-insensitive surfaces.We present in vivo evidence, including human tumors, that the two macroH2A1 isoformsshow cell-type specific expression, in particular with regard to proliferation. Could there bemetabolic control of gene activity through macroH2A-containing chromatin and how is thislinked to proliferation and gene repression in mammals?

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Hyockman Kwon Abstract P65BAF53-dependent higher-order chromatin structure as thecompartment of replication and repair foci

Ki Won Lee1, Su Jin Kwon1, Phan Kyu Park1, Jae Yong Kim1, Yunhee KimKwon2, and Hyockman Kwon1

1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies,Yongin 449-791, Republic of Korea, 2Department of Biology, Kyunghee University, Seoul130-701, Republic of Korea

It is becoming evident that higher-order chromatin structure plays a critical role in manyaspects of gene regulation in interphase mammalian nuclei. The emerging view is thatchromosomes are compartmentalized into discrete chromosome territories in whichchromatins are packaged into compact chromosomal subdomains with diameters of 100 to450 nm. Chromosomal subdomain may define the functional compartment as well as thestructural compartment. In mammalian cell nucleus, DNA replication and DNA double-strand break repair occur at discrete sub-nuclear structure called replication foci and repairfoci, respectively. Interestingly, the average sizes of replication and repair foci, ~1 Mb, aresimilar each other, suggesting that they could represent different functional states ofchromosomal subdomains. However, this possibility has not been fully appreciated yet.Previously, we showed that BAF53 is required for the higher-order chromatin structure.BAF53 knockdown resulted in the expansion of chromosome territories and the remarkableincrease in the micrococcal nuclease sensitivity of chromatin. Here we found that BAF53knockdown suppressed the formation of replication foci and repair foci. Although DNAreplication proceeded normally in the BAF53-knockdowned cells, the early S-phasereplication foci were not observed. Instead, a diffused pattern of BrdU incorporation wasfound in the nucleoplasm. Interestingly, the mid and late S-phase replication foci remainedintact. Reduction of H3-K9 dimethylation foci in the nucleoplasm in the BAF53-knockdowned cells supports the specific disappearance of the early S-phase replicationfoci. In addition, the formation of H2AX foci in response to DNA damage by adriamycin waslargely reduced in the BAF53-knockdowned cells. Activation of ATM appeared unchanged inthe BAF53-knockdowned cells. Taken together, these results raised the possibility thatreplication foci and repair foci are originated from the same structural entity such aschromosomal subdomain whose formation requires BAF53. We discussed our results basedon the multi-loop subcompartment model for chromosomal subdomain.

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Monika Lachner Abstract P66Studying lysine methylation in non-histone proteins

Monika Lachner1, Kristie L. Rose2, Jeffrey Shabanowitz2, Karl Mechtler3,Thomas Jenuwein3, Donald F. Hunt3 and C. David Allis1

1The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A., 2Department ofChemistry, University of Virginia, Charlottesville, VA 22901, U.S.A., 3Research Institute ofMolecular Pathology, Dr. Bohrgasse 7, 1030 Wien, Austria

While the impact of histone lysine methylation on chromatin structure and function has beenextensively studied, there is little known about the occurrence and function of this post-translational modification in non-histone proteins. This obvious lack of understandingsparked our interest in exploring lysine methylation in non-histone proteins.In order to identify novel lysine methylation sites in non-histone proteins, we employed anapproach that combines immunoprecipitation with a pan-methyl-lysine antibody and mass-spectrometry. These experiments provided several interesting candidate proteins that areinvolved in a variety of cellular processes. For a more detailed analysis we chose two ofthese proteins (Eset and mAM), since they have a well-documented function. Eset andmAM form an enzymatic complex that specifically trimethylates histone H3 on lysine 9.Interestingly, immunoprecipitations with the pan-methyl-lysine antibody suggested that bothcomplex members are lysine-methylated proteins. At this point, we have confirmed thisfinding and we have been able to map one methylation site in mAM. Experiments arecurrently underway that will address the identity of the respective methyltransferase as wellas the potential functional implications of this post-translational modification (e.g.modulation of the enzymatic activity of the complex, regulation of protein-proteininteractions).We anticipate that further investigation of lysine methylation in non-histone proteins willdemonstrate the importance of this post-translational modification in numerous biologicalprocesses.

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Brian D. Larsen Abstract P67Caspase 3 mediated DNA strand breaks contribute togenomic reorganization during skeletal muscle terminaldifferentiation

Brian D. Larsen1,2 and Lynn A. Megeney1,2

1Ottawa Health Research Institute, Centre for Stem Cell Research, Molecular MedicineProgram, Ottawa Hospital General Campus, Ottawa ON, Canada. 2Department of Cellularand Molecular Medicine, University of Ottawa, Ottawa ON, Canada

Skeletal muscle differentiation is dependent on chromatin remodelling and genome widereorientation of gene expression programs. However, the factors controlling this transitionhave not been fully elucidated. One mechanism known to influence the differentiationprocess is the activity of pro-apoptotic proteins. The apoptotic process is itself associatedwith genomic reprogramming events i.e. DNA strand breaks, suggesting that similarmechanisms may influence the genomic transition during cell differentiation. Here weexplored the role of strand break formation in skeletal muscle differentiation. In situ nicktranslation (ISNT) was used to detect DNA strand breaks during differentiation of an in vitromodel system (C2C12 myoblast cell line). Transient DNA strand breaks were detected earlyduring differentiation between 12 and 24 hours. The formation of DNA strands breakscoincided with caspase 3 activation, a requirement for skeletal muscle differentiation.Moreover, treatment of differentiating C2C12 myoblasts with the caspase 3 inhibitor (z-DEVD-fmk) blocked the detection of DNA strand breaks by ISNT coincident with aninhibition of differentiation/myotube formation. A well characterized function of caspase 3 isthe activation of the caspase activated nuclease (CAD) through proteolytic cleavage of itsinhibitor (ICAD). Western blot analysis revealed cleavage of the long isoform of ICAD inC2C12 cells at 12 and 24 hours following low serum induction of differentiation; theseresults suggest the stand breaks are mediated through caspase 3 activity. The formation oftransient DNA strand breaks implicates the necessity of a DNA repair mechanism duringthe differentiation process. As such we monitored the phosphorylation status of the histonevariant H2AX, a well characterized marker of DNA double strand breaks, during myoblastdifferentiation. Phosphorylation of H2AX coincided with the formation of DNA strandbreaks during myoblast differentiation and also appeared to be dependent on caspase 3activity. These results suggest a role for pro-apoptotic proteins in regulating gene expressionand chromatin remodelling to promote myoblast differentiation.

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Richard Lawrence Abstract P68Mechanisms controlling dynamic Swi6/HP1 binding in S.pombe facilitate de novo heterochromatin formation

Richard J. Lawrence and Thomas A. Volpe

Department of Cell and Molecular Biology, Feinberg School of Medicine, NorthwesternUniversity, 303 E. Chicago Avenue, Chicago, IL 60611, U.S.A.

Heterochromatin plays a critical role in genome stability and organization, and has longbeen regarded as statically condensed and inert. Contrarily, recent evidence suggests thatheterochromatin is maintained by dynamic binding of heterochromatin protein 1 (HP1).However, the mechanisms, if any, that mediate dynamic HP1 binding and the in vivofunction of HP1 mobility are unknown. We are studying a jmjC domain protein thatmodulates binding of the HP1 homolog, Swi6, to heterochromatin in S. pombe therebyantagonizing epigenetic stability. Interestingly, the protein lacks histone demethylase activity.In the absence of this protein a reporter gene in centromere heterochromatin is moreenriched with Swi6 and in vivo binding studies indicate that Swi6 is more tightly bound toheterochromatin. Paradoxically, this “hyper-heterochromatization” promotes epigeneticheterochromatin stability but inhibits de novo heterochromatin nucleation. These resultsultimately suggest that Swi6 binding is actively antagonized by this protein; thus, it functionsto mobilize a pool of Swi6 that targets heterochromatin nucleation, most likely through anovel mechanism that is independent of histone demethylase activity.

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Frederic Leduc Abstract P69Presence of gamma-H2AX in elongating spermatids:involvement of NHEJ?

Frederic Leduc, Veronique Lauziere, Marilyne Joly, Leila Jaouad andGuylain Boissonneault

Faculte de Medecine, Departement de Biochimie, 3001, 12eme avenue Nord, Sherbrooke,Qc J1H 5N4, Canada

The histone variant H2AX is involved in early DNA damage response and controls therecruitment of DNA repair proteins at sites of double-stranded breaks. In testis, the active,phosphorylated form of H2AX (gamma-H2AX) is thought to be involved in two processesnamely the inactivation of sex chromosomes (at the sex vesicle) and the control of genomeintegrity during meiotic recombination. We used confocal microscopy applied to bothimmunofluorescence and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-endlabeling) on squash preparations of seminiferous tubules. Stage-specific sections wereobtained according to their light absorption pattern. Here, we demonstrate the presence ofgamma-H2AX in the whole population of elongating spermatids in mouse (steps 8-9),coincident with the onset of transient DNA strand breakage and chromatin remodeling asshown by the hyperacetylation of histone H4. These results strongly suggest that acomplex DNA repair system is recruited and required during the chromatin remodeling stepsin elongating spermatids. Given their haploid character, we hypothesize that the non-homologous end-joining (NHEJ) is responsible for the DNA repair during step 8 through 13.The presence of NHEJ-associated factors is being investigated.Funded by Canadian Institutes of Health Research (Grant# MOP-74500)

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Min Gyu Lee Abstract P70Functional association of a trimethyl H3K4 demethylase andRing6a/MBLR, a polycomb-like protein

Min Gyu Lee1, Jessica Norman1, Anne llvarsonn2, Joel C Eissenberg2, AliShilatifard2 and Ramin Shiekhattar1

1The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 U.S.A., 2Department ofBiochemistry and Molecular Biology, Saint Louis University Health Sciences Center, SaintLouis, MI 63104, U.S.A.

Histone methylation is a post-transcriptional mark regulating chromatin structure and generegulation. Once deemed irreversible, recent findings have identified two classes ofenzymes capable of demethylating lysine residues. BHC110/LSD1, the catalytic heart ofmultiple co-repressor complexes, was the first of such demethylases shown to reversedimethyl histone H3K4. However, due to intrinsic limitations of BHC110/LSD1 mode ofaction, it is unable to remove trimethyl H3K4 marks. Here we show that JARID1d, a memberof a second class of histone demethylases containing JmjC-domain, can specificallydemethylate trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjC-domain, the BRIGHT and zinc-finger-like C5HC2 domains are required for maximumcatalytic activity. Importantly, isolation of native JARID1d complexes from human cellsrevealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR.Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymaticactivity. We show that JARID1d occupies human Engrailed 2 promoter and regulates itsexpression and H3K4 methylation levels. Finally, we show that the single Drosophilahomolog of JARID1d, little imaginal discs (Lid), is also a trimethyl H3K4 demethylaseattesting to the cross-species conserved function for this family of histone demethylases.

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Niraj Lodhi Abstract P71Histone acetylation (H3K9) and methylation (H3K4) of thenucleosome over core promoter are associated with theinduction of tobacco PR-1a gene

Niraj Lodhi, C. P. Chaturvedi, Suraiya A. Ansari, Rakesh Srivastava,Samir V. Sawant and Rakesh Tuli

National Botanical Research Institute India, Rana Pratap Marg, Lucknow, India 226001

The PR-1a gene encodes one of the major defense related protein in tobacco (Nicotianatabacum) and it is tightly regulated at the transcription level. Detailed studies on PR-1apromoter in tobacco leaves have shown that it is induced specifically on pathogen attack orafter Salicylic Acid (SA) induction. Our work suggested that the core promoter region of PR-1a gene plays an important role in determining SA induced transcription. We have mappeda nucleosome which spanned the core promoter of PR-1a gene. The nucleosome spannedthe region between -103 to +55 relative to the transcription initiation site of the PR-1a gene.We carried out Chomatin Immunoprecitation (ChIP) with H3K9 acetylation specific andH3K4 methylation specific antibodies to identify the role of acetylation and methylation ofnucleosome over the core promoter region. Our results revealed that the nucleosome overthe core promoter results in a repressive chromatin architecture which is remodeled byhistone modifications concomitant with the gene activation after SA induction. The SAinduction leads to shifting of nucleosome from the core promoter which allows the assemblyof the pre-initiation complex and initiation of transcription. The results related to our newfindings will be presented at the conference.

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Mattias Mannervik Abstract P72An HDAC3/SMRTER/Ebi complex required for Snailrepressor function in Drosophila development

Mattias Mannervik, Dai Qi and Mattias Bergman

Stockholm University, Wenner-Gren Institute, Dept. of Developmental Biology,Arrheniuslaboratories E3, Stockholm, Sweden

The zinc-finger transcription factor Snail is critical for Drosophila embryo development bypreventing expression of neuroectoderm-specific genes in the mesoderm. We found that inembryos devoid of maternal Ebi protein, Snail repressor function is impaired. Drosophila Ebiand its mammalian homolog TBL1 are WD40 proteins with a divergent F-box domain.Previous studies have linked Ebi and TBL1 to two different pathways; ubiquitin conjugationthrough SCF-type ligases, and N-CoR/SMRT/HDAC3-mediated transcriptional repression. Inebi mutant embryos, Snail target genes are de-repressed in the mesoderm. De-repressionof a Snail-dependent reporter gene in ebi mutant embryos, and genetic interactions withsnail support a requirement for Ebi in Snail function. Snail-mediated repression waspreviously shown to depend on another co-repressor, CtBP. We found that both CtBP andEbi can interact with Snail protein in vitro, but through different interaction domains. Aminimal Ebi-interaction domain that fails to bind CtBP constitutes a potent repressiondomain in both S2 cells and in transgenic embryos. This suggests that Snail uses Ebi as co-repressor independently of CtBP. The repression activity of this domain can be attenuatedeither by knockdown of HDAC3 or by TSA treatment, indicating an involvement of histonedeacetylation. By contrast, inhibition of proteasome activity does not affect Snail-mediatedrepression. We suggest that Ebi as part of a SMRTER/HDAC3 co-repressor complex isrequired for Snail function in Drosophila, and that histone deacetylation is part of themechanism by which Snail represses transcription.

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Robert Martin Abstract P73Chromatin labeling and distribution in living cells

Martin, R.M., Leonhardt, H. and Cardoso, M.C.

Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin,Germany and Ludwig Maximilians University Munich,Department of Biology II, 82152Planegg-Martinsried, Germany

Live cell fluorescence microscopy experiments often require visualization of the nucleus andthe chromatin to determine the nuclear morphology or the localization of nuclearcompartments. We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide,Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuousimaging and photobleaching in widefield epifluorescence and confocal laser scanningmicroscopy. In addition, we compared the DNA stainings with fluorescent histones as anindependent fluorescent label to mark chromatin. From the dyes tested, only Hoechst andDRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had severaladvantages, namely low photobleaching, labeling of the chromatin compartmentscomparable to that of H2B-GFP fusion proteins, and deep red excitation/emissioncompatible with available genetically encoded fluorescent proteins such as C/G/YFP ormRFP. The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and caneasily be combined with other fluorophores with blue to orange emission. It could be used ina variety of cells of different species including primary cultures after a few minutesincubation in the culture medium.Furthermore the effect on histones and other chromatin proteins will be discussed. DRAQ5is a useful molecular tool for cell biology that allows a fast and non invasive labeling as wellas microscopic visualization of DNA structures in living cells.

Ref:Martin, R. M., Leonhardt, H., and Cardoso, M. C. (2005) Cytometry A 67, 45-52

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Peter McKeown Abstract P74Chromatin components of the Arabidopsis thaliana nucleolus

Peter C. McKeown, Alison F. Pendle and Peter J. Shaw

Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park,Colney Lane, Norwich, Norfolk U.K., NR4 7UH, John Innes Centre, Colney Lane, Norwich,Norfolk, NR4 7UH, U.K.

Eukaryote DNA is packaged into chromatin through association with highly basic histoneproteins. The presence of histone variants and multiple covalent modifications engender a‘histone code’ which controls the expression of the packaged DNA. This chromatin is notpurely informational – it also has a structural existence within the nucleus. This is well-demonstrated in the largest nuclear structure, the nucleolus, which forms through thechromatin-regulated expression of rRNA genes and acts as the site of pre-ribosomeformation and many RNA processing pathways. However, few distinctive chromatin markshave been identified in the nucleolus, in contrast to other structures such as centromeres,telomeres and sites of DNA breaks.We have now identified a group of nucleolar histones and histone modifications in themodel plant, Arabidopsis thaliana thaliana, through a combination of techniques. Previously,we used mass spectrometry of extracted nucleoli to analyse the organellar proteome(Pendle et al., Mole. Biol. Cell, 16:260-269, 2005) identifying several nucleolar histones inthe process. We have now used further MS techniques to determine how these proteins aremodified, and demonstrated nucleolar specificity with GFP-fusion proteins.Immunofluorescence has identified other modifications, and confirmed the presence of anucleolus-specific linker histone which may bind inactive rRNA genes and acetylatedhistone H2B which colocalises with sites of RNA polymerase I-mediated transcription. Suchchromatin components may represent a source of nucleolus-specific information additionalto the standard histone code.We conclude that the nucleolus is a good model for assessing the links between theinformational content of the histone code and the its effects upon nuclear structures, andare currently determining the roles of the chromatin marks identified in both rRNAtranscription and nucleolar structure.

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Rosalind Meldrum Abstract P75Visualisation of DNA repair and chromatin dynamics

Rosalind Meldrum

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT U.K.

The area of research describing interactions between chromatin structure and the DNAdamage response is receiving an increasing amount of attention. It is likely that DNAdamage has a considerable influence on the physical structure and movements ofchromatin. It is with out doubt that new high-resolution microscopy techniques, that canvisualise detailed geometrical and spatial changes that take place in the cell in response toDNA damage, will play a critical role in this area of research.The most useful approaches to detecting and visualising chromatin dynamics are thosewhere DNA damage can be induced in predetermined patterns and target specificstructures in a cell nucleus.To be able to see the details of structural and dynamic changes it is desirable to induce andvisualise DNA damage with very high resolution.UV lesions can be induced with nanoscale resolution in cell nuclear DNA by triple-photoninfra-red absorption. The number or lesions induced is much smaller than when total cellirradiation by a mercury lamp is used or when localised damage is induced by irradiation ofcells with UV light through a micro-filter. Because the UV photoproducts can be induced in adefined pattern, during a period of incubation following irradiation, distinctive movement ofthe damage DNA is seen to take place and the damage distributed over the cell nucleusforms individual clusters of lesions. The characteristics of the clustering of lesions are beinginvestigated in relation to cell cycle, histone modification, ATP-dependent remodelling andincorporation of histone variants. A further interesting observation revealed that higherlevels of damage immobilise and preserve the induced pattern for some hours followingirradiation.

Ref:R.A.Meldrum, S.W.Botchway, C.W.Wharton, G.J.Hirst. (2003) Nanoscale induction of UVphotoproducts in cellular DNA by 3-photon near infra-red absorption EMBO Reports 4, 121144-1149

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Brendon Monahan Abstract P76Purification and characterization of the fission yeast Swi/Snfand RSC chromatin remodeling complexes

Brendon J. Monahan1, Judit Villen2, Samuel Marguerat3, Jurg Bahler3,Steve Gygi2 and Fred Winston1

1Department of Genetics, Harvard Medical School, Boston MA, U.S.A, 2Taplin BiologicalMass Spectrometry Facility, Department of Cell Biology, Harvard Medical School, BostonMA, U.S.A., 3Wellcome Trust Sanger Institute, Cambridge, U.K.

The Swi/Snf type of ATP-dependent chromatin remodeling complexes are molecular motorsthat modify chromatin structure thereby regulating transcription and mediating other cellularprocesses such as DNA repair. Two evolutionarily conserved and distinct subclasses ofSwi/Snf, named Swi/Snf and RSC, have been extensively studied in Saccharomycescerevisiae. Here we present the purification and characterization of the Swi/Snf and RSCchromatin remodeling complexes from the fission yeast Schizosaccharomyces pombe. S.pombe, which is as closely related evolutionary to humans as it is to S. cerevisiae, is ofinterest as its chromatin shares several important similarities to mammalian chromatin thatdo not occur in S. cerevisiae and also provides a basis for valuable comparative analysis.The S. pombe Swi/Snf and RSC complexes were purified using tandem affinity purification(TAP) methodology and components identified by mass spectrometry. The S. pombeSwi/Snf complex is composed of 12 subunits, of which six are shared with the 14-memberRSC complex. Deletion and tetrad analysis has shown that the core subunit genes in RSCare essential for growth whereas deletion mutants of the paralogous Swi/Snf genes wereviable. Four of the six genes shared between the two complexes are essential for cellgrowth, the exceptions being the two arp (actin related protein) genes, arp4+ and arp9+. Toinvestigate the global effect Swi/Snf has on S. pombe gene expression, whole genomeexpression analysis was done using deletion mutants of two core Swi/Snf complex subunitgenes, snf22 and snf5. Overall, the expression levels of approximately 2.5% of S. pombegenes were altered greater than 2-fold in the swi/snf mutants. Interestingly, genes involvedin sugar uptake and iron homeostasis were significantly enriched in the up-regulated geneset. This work has established the foundation for further detailed analysis into the role ofthese chromatin remodeling complexes in transcription activation and repression and othercellular processes in fission yeast.

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Antonin Morillon Abstract P77Transcriptional co-suppression in S. cerevisiae

Julia Berreta, Benjamin Pernot-Cornu and Antonin Morillon

CGM-CNRS, Gif/Yvette, France

Cosuppression has been defined as high gene copy number-triggered, homology-dependent, gene silencing and may have evolved in eukaryotic cells as a defensive strategyagainst viral infections and to control activity of transposons. In S. cerevisiae, co-suppression was first unveiled in 2002 (Y. Jiang, 2002) and was shown to act at thetranscriptional level controlling the Ty1 retrotransposon. As the main actors ofTranscriptional Gene Silencing are not conserved in baker yeast, co-suppression must bemediated by an original pathway which we try to characterize in this work. We hypothesizethat it is mediated by regulatory RNAs which may inhibit Ty1 expression. We show that theexpression of Ty1 is dependent upon the presence of several proteins involved in RNAdegradation, in particular the 5-3 exonuclease Xrn1. Furthermore, the drop of Ty1expression correlates with the accumulation of an antisense non coding RNAcorresponding to the 5 Long Terminal Repeat (LTR) region of Ty1. Through ChIPexperiments, we show that the decrease of Ty1 RNA does not correlate to a reduction ofRNAPII occupancy on the Ty1 gene suggesting that Ty1 RNA is affected at a post-transcriptional step. Finally, it has been shown that the retrotransposon is very sensitive tothe histone levels and to Chromatin Remodeling Complex activity. We will discuss ourpreliminary results on nucleosome positions and histone modifications on Ty1 elementsupon co-suppression conditions.

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Ashby Morrison Abstract P78Mec1/Tel1-dependent phosphorylation of a chromatinremodeling complex influences the DNA damagecheckpoint pathway

Ashby J. Morrison1, Jung-Ae Kim2, Maria D. Person3, Jessica Highland1,Tammy S. Wehr1, Sean Hensley1, Jianjun Shen1, Sean R. Collins5, JeffDelrow4, Nevan J. Krogan5, James E. Haber2 and Xuetong Shen1

1Department of Carcinogenesis, Science Park Research Division, University of Texas M.D.Anderson Cancer Center, Smithville, Texas 78957, 2Rosentiel Center and Department ofBiology Brandeis University, Waltham, Massachusetts 02454, 3College of Pharmacy,Division of Pharmacology & Toxicology, University of Texas at Austin, Austin, Texas 78712,4Division of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, Washington98109, 5Department of Cellular and Molecular Pharmacology, University of California SanFrancisco, San Francisco, California 94143 U.S.A.

The Mec1/Tel1 kinases in yeast, ATM/ATR in mammals, play central roles in coordinatingthe DNA damage response by phosphorylating proteins involved in DNA repair andcheckpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such asthe yeast INO80 complex, which were originally characterized as transcriptional regulators,have also been implicated in the DNA damage response. Here, we show that the Ies4subunit of the INO80 complex is phosphorylated in a Mec1/Tel1-dependent manner duringexposure to DNA damaging agents. The phosphorylation status of Ies4 does notsignificantly affect transcription or DNA repair processes, such as homologousrecombination. However, DNA damage checkpoint pathways are influenced by thephosphorylation status of Ies4. These findings establish a chromatin remodeling complexas a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulatescheckpoint responses, and suggest that post-translational modification of chromatinremodeling complexes may regulate their involvement in distinct nuclear processes.

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Raul Mostoslavsky Abstract P79Genomic instability and aging-like phenotype in the absenceof mammalian SIRT6

Raul Mostoslavsky1, David B. Lombard1,2, Katrin F. Chua1, Jennifer Kim1,Lionel Gellon4, Bruce Demple4, George Yancopoulos3 and Frederick W. Alt1

1Howard Hughes Medical Institute, The Children’s Hospital, CBR Institute for BiomedicalResearch, and 2Department of Genetics, Harvard Medical School, Boston, Massachusetts021151, 3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts021152, 4Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NewYork 10591-67073, 5Department of Genetics and Complex Diseases, Harvard School ofPublic Health, Boston Massachussets 021154

In yeast, the histone deacetylase/ADP-ribosyltransferase Sir2 inhibits DNA recombination,promoting longevity. Seven mammalian Sir2 homologs, termed SIRT1-SIRT7, have beendescribed. Here we show that SIRT6 is a nuclear, chromatin-associated protein, expressedin multiple tissues. At the cellular level, SIRT6 promotes resistance to DNA damage andsuppresses genomic instability, in association with a role in Base Excision Repair (BER).SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that includeacute lymphocyte depletion, loss of subcutaneous fat, lordokyphosis and severe metabolicdefects, eventually dying at about 4 weeks.We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6loss in mice leads to defects in lymphocyte homeostasis and abnormalities that overlap withaging-associated degenerative processes. Recent progress in understanding the role ofSIRT6 in DNA repair and metabolism will be discussed.

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Takahiro Nakayama Abstract P80Drosophila GAGA factor promotes histone H3.3 replacementthat prevents the heterochromatin spreading

Takahiro Nakayama, Kenichi Nishioka, Yi-Xin Dong, Tsukasa Shimojimaand Susumu Hirose

Department of Developmental Genetics, National Institute of Genetics, and Department ofGenetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan

Drosophila white gene that is normally located euchromatin region governs a red eyephenotype. When the white gene is juxtaposed with centromeric heterochromatin regions bychromosomal rearrangement, its expression is subject to variable in a heritable silencingmanner, giving rise to white mottled eye color. This phenomenon termed position effectvariegation (PEV) provides evidence for a crucial role of chromatin structure in geneexpression. Previously, Karch group showed that the Trithorax-like gene encoding GAGAfactor is a dominant enhancer of PEV, suggesting that the GAGA factor plays a role in theactive maintenance of white under heterochromatin environment. However, little is knownabout the molecular mechanism. Here, we demonstrate that the GAGA factor binds to a sitejust downstream of the white gene and this binding site is necessary and sufficient to blockheterochromatin spreading. Interestingly there are a dip of histone H3 Lysine 9 methylationand a peak of H3 Lysine 4 methylation at this site. Furthermore, the GAGA factor promoteschromatin remodeling and replacement of histone H3 with H3.3 through recruitment ofHIRA at this site, and maintains white expression under the heterochromatin environment.Based on these findings, we propose that the GAGA factor- dependent replacement ofLysine 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin.

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Zuyao Ni Abstract P81The tumor suppressor BRG1 silences the distal silencers atinterferon-responsive genes

Zuyao Ni*, Mohamed A I Abou El Hassan*, Zhaodong Xu, Tao Yu, andRod Bremner

Toronto Western Research Institute, University Health Network, 399 Bathurst Street,Toronto, Ontario, M5T 2S8, Departments of Lab Medicine and Pathobiology andOphthalmology and Vision Science, University of Toronto, Ontario, Canada

Communication between distal regulatory DNA elements is essential for control of manynuclear processes in higher eukaryotes. Long-range gene regulation has been associatedprimarily with step-wise activation of differentiation genes over days. Recently, our groupimplicated long-range effects in rapid IFNgamma (IFNg)-mediated gene induction. However,the mediators of such long-range effects are unknown. Previously, we showed that thetumor suppressor and chromatin remodeling factor BRG1 has a primary role in regulatingIFNg-responsive genes, so we hypothesized it may act through remote elements. Here, weshow that BRG1 constitutively binds distal regulatory sites and binding increases upon IFNgtreatment. BRG1 is required for transcription factor (STAT1 and IRF1) binding, histoneacetylation, and chromatin remodeling at the distal IFNg sensitive sites at several target loci. At both CIITA and SOCS1 loci distal elementsloop and interact with target promoters and/or with each other in a BRG1 dependentmanner. To determine the functional relevance of these events in the appropriate context,we constructed a 200 kb bacterial artificial chromosome (BAC) reporter vector containingthe entire CIITA locus. Remarkably, deleting any one of several distal elements inducedbasal CIITA activity to levels similar to those seen in IFNg-treated cells, even in the absenceof BRG1. Thus, multiple remote elements cooperate to silence CIITA, and IFNg-overcomestheir effect, derepressing CIITA in a BRG1-dependent manner. Negative regulation of distalsilencing complexes is a novel mechanism of action of BRG1.

(* Equal contributors)

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Olivia Osborn Abstract P82Transcriptional targets of Af4

Osborn O, Oliver P.L., Bitoun E. and Davies K.E.

Dept of Physiology Anatomy & Genetics, Oxford University

The putative transcription factor Af4, known to be translocated in acute leukaemia, is alsoexpressed in the Purkinje cells of the cerebellum. The robotic mouse mutant has an ataxicgait, is smaller than its littermates and shows distinct and progressive pattern of Purkinjecells loss from 8 weeks of age.This phenotype is caused by a gain of function mutation (V280A) in Af4 which prevents itsnormal rapid turnover by the proteosome and consequently its accumulation in numeroustissues as well as the brain. The cause of the cell loss in robotic and the normal function ofAf4 is, however, unknown.Chromatin immunoprecipitation coupled with microarrays have been used with the aim offinding the direct targets of this transcription factor along with expression analysis todetermine the downstream targets that contribute to the ataxic phenotype.

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Julia Pagan Abstract P83A novel corepressor, BCOR-L1, functions through CTBP andclass 2 HDACs

Julia K. Pagan1,2, Jeremy Arnold1, Kim J. Hanchard1, Mathew J.K. Jones1,2,Derek J. Richard1, Alistair Forrest3, Amanda Spurdle1, Eric Verdin4, MerlinCrossley5, Georgia Chenevix- Trench1, David B. Young*1 and Kum KumKhanna*1

1Queensland Institute of Medical Research, 300 Herston Rd, Herston 4029, Queensland,Australia, 2School of Medicine, Central Clinical Division, University of Queensland, RoyalBrisbane Hospital, Herston 4029, Queensland, Australia, 3Institute for Molecular Bioscience,University of Queensland, Brisbane, QLD 4072, Australia, 4Gladstone Institute of Virologyand Immunology, University of California San Francisco, San Francisco, California, UnitedStates of America, 5School of Molecular and Microbial Biosciences, G08, University ofSydney, New South Wales 2006, Australia

Corepressors play a crucial role in negative gene regulation and are defective in severaldiseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe andfunctionally characterize BCoR-L1, a homolog of BCoR corepressor. When tethered to aheterologous promoter, BCoR-L1 is capable of strong repression. Most corepressorsfunction by associations with histone deacetylase (HDAC) activity. BCoR-L1 coprecipitateswith Class II HDACs; HDAC4, HDAC5 and HDAC7, suggesting they are involved in its roleas a transcriptional repressor. BCoR-L1 interacts with the CtBP corepressor through aCtBP-interacting motif in its amino-terminus. Abrogation of the CtBP binding site withinBCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore,BCoR-L1 is located on the E-Cadherin promoter, a known CtBP regulated promoter, and isinvolved in repression of E-Cadherin.

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Maria V. Panchenko Abstract P84Role of Jade-1 in the HAT HBO1 complex

Rebecca L. Foy, Vipul C. Chitalia, Herbert T. Cohen and Maria V. Panchenko

Boston University School of Medicine, Evans Biomedical Research Center, MA 02118-2393, U.S.A.

Regulation of global chromatin acetylation is important in processes requiring chromatinremodeling, including DNA replication. We demonstrated that PHD zinc finger protein Jade-1 is localized to the nucleus, activates rates of transcription when tethered to aheterologous promoter, and is associated with endogenous HAT activity. It has been recentlyreported (J. Coté’s lab, 2006) that Jade-1 co-purifies with a novel HAT complex, consistingof three additional proteins, including HBO1, ING4/5 and Eaf6. In mammalian cells, HBO1provides the basal level of global histone H4 acetylation, is required for DNA replication,and its activity is regulated during the cell cycle. We thus investigated a functional role forJade-1 in the HBO1 complex. We demonstrated that while overexpression of HBO1 did notalter levels of endogenous histone H4 acetylation, co-transfection of even low sub-sufficientamounts of Jade-1 resulted in a dramatic, up to a 50-fold upregulation of histone H4acetylation, strongly suggesting that Jade-1 plays a crucial role in HBO1-mediatedacetylation of nucleosomal histones. Interestingly, while PHD fingers were indispensable forJade-1 to synergize with HBO1, they were dispensable for Jade-1-HBO1 physicalinteractions. We proposed that Jade-1 might promote histone acetylation by bindingchromatin via its PHD fingers and targeting the HAT HBO1 complex to the proximity ofhistone substrates. Because Jade-1 partner HBO1 is involved in DNA replication, weinvestigated an effect of cell cycle progression on endogenous Jade-1 expression andnuclear distribution. We found that Jade-1 nuclear distribution and presumably chromatinassociation is strongly regulated by cell growth arrest and during cell cycle progression. Inaddition, Jade-1 undergoes phosphorylation followed by dephosphorylation in synchronizedcycling cells. The data suggest a role for cdks in this posttranslational modification of Jade-1 and implicates a potential mechanism for the regulation of HBO1 HAT activity during thecell cycle and DNA synthesis.

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Tej Pandita Abstract P85Mammalian ortholog of Drosophila MOF is critical forembryogenesis and DNA repair

Arun Gupta1, Geraldine Guerin-Peyrou2, Girdhar G. Sharma1, ManjulaAgarwal1, Raj K. Pandita1, Raju Kucheralapati3, Thomas Ludwig2 andTej K. Pandita1

1Washington University School of Medicine, Saint Louis, MO 63108; 2College of Physiciansand Surgeons, Columbia University, New York, NY 10032, 3Harvard Medical School, Boston,MA 02115, U.S.A.

Human ortholog (hMOF) of the Drosophila MOF gene (males absent on the first) is histoneH4 lysine K16-specific acetyltransferase. It is involved in transcription as it is a component ofa functional dosage compensation complex required for male killing in Drosophila and DNAdamage response. Cellular exposure to ionizing radiation enhances hMOF-dependentacetylation of its target substrate, lysine 16 of histone H4. Depletion of hMOF results inabrogation of ATM autophosphorylation, ATM kinase activity and DNA repair as well asincreases cell killing after IR exposure. Based on these preliminary studies, we hypothesizethat hMOF is involved in the regulation of DNA damage-induced ATM activation. In addition,MOF function is indispensable for development, because Mof-deficiency in mouse embryosresults in early embryonic lethality which cannot be overcome by inactivation of p53 or ATM.Mice with haploinsufficiency of mMof in Atm null background are smaller in size and dieearly mostly because of leukemia. MOF over expression results in enhanced oncogenictransformation and is over expressed in most of the human tumors. These resultsdemonstrate that ‘MOF’ is not only required for early embryonic development but plays acritical role in cell growth during oncogenic transformation in absence of Atm. We willdiscuss the role of MOF in regulation of DNA double strand break repair.

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Maëlle Pannetier Abstract P86Imprinting perturbation in mouse hepatocarcinoma: linkbetween DNA methylation and histone methylation

Maëlle Pannetier, Katia Delaval, Alexandre Wagschal and Robert Feil

Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535 et Université MontpellierII, 1919 route de Mende, 34293 Montpellier cedex 5, France

Genomic imprinting is an essential mechanism in development that gives rise to mono-allelicexpression of genes depending on the parental origin of the allele. This mono-allelicexpression is controlled by imprinting control regions (ICRs), short CpG-rich sequences thatare differentially methylated. In addition to differential DNA methylation, allelic histonemodifications, like lysine methylation and acetylation, are observed. We found thattrimethylation of H3K27, H3K9 and H4K20 are associated with the DNA-methylated allele,whereas dimethylation of H3K4 and acetylation of H3 are associated with the unmethylatedone. Cancer cells are characterized by prominent epigenetic dysregulation, including alteredDNA methylation patterns, chromatin modifications and loss of genomic imprinting. Using amurine model of hepatocarcinoma, we study such epigenetic alterations at two imprinteddomains located on chromosome 7: the Kcnq1 and Igf2/H19 domains. Transgenic miceshowing liver-specific expression of a c-myc transgene were crossed with P53 knock-outmice. Thus, c-myc overexpression, in a hemizygous state for P53, gives rise to liver tumoursat about 9 months of age. Moreover, these mice have a paternal chromosome 7 from M.Spretus and a maternal one from M. Domesticus, allowing us to discriminate the maternalallele from the paternal one. In this context, we determine if imprinting is perturbed in thus-induced hepatocarcinomas, studying DNA methylation and histone modifications. 1/4 oftested tumours showed a gain of DNA methylation at the Igf2/H19 ICR, whereas 1/16showed a loss of DNA methylation at the Kcnq1 ICR. Analysis of histone modifications byChIP will allow us to determine how DNA and histone modifications are correlated duringepigenetic perturbation.

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Janet Partridge Abstract P87Establishment and maintenance of centromericheterochromatin in fission yeast are functionally separable

Janet F. Partridge, Jennifer L. DeBeauchamp, Michael Hadler, Dagny L.Ulrich and Victoria J.P. Noffsinger

Dept. Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN. U.S.A.

Both the establishment and maintenance of centromeric heterochromatin in fission yeastrequire the RITS complex. Comprised of centromeric siRNAs, the chromodomain proteinChp1, Argonaute (Ago1) and Tas3, RITS couples the cellular RNAi pathway with assemblyof constitutive heterochromatin. However, it remains unclear if mechanisms governing RITS-dependent establishment of centromeric heterochromatin differ from its maintenance. Here,we generate a Tas3 protein, mutated in a highly conserved GW-rich Argonaute-bindingdomain, which cannot bind Ago1. This mutant exhibits near normal maintenance ofcentromeric heterochromatin, but cannot support its establishment. We show that Ago1 canbe maintained at centromeres through binding siRNA, but to establish centromericheterochromatin, Ago1 must bind Tas3-Chp1. Our results support a model wherebyrecruitment of RITS to centromeres is initiated by Chp1 binding to K9-methylated histoneH3, with the RNAi pathway responsible for maintenance of RITS at centromeres.

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Kelly Perkins Abstract P88Activated HIV-1 provirus forms a gene loop, connecting viraltranscriptional initiation with termination

Kelly J. Perkins, Marina Lusic2, Mauro Giacca2 and Nick J. Proudfoot1

1Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford OX13RE, U.K., 2Laboratory of Molecular Medicine, International Center for Genetic Engineeringand Biotechnology (ICGEB), Padriciano, 99-34012 Trieste, Italy

We are investigating high order chromatin structure and factor recruitment to integrated HIV-1 provirus following re-activation from latency. Both chromosome conformation capture (3C)and chromatin immunoprecipitation (ChIP) techniques are being utilized. 3C analysis of pro-monocytic cell line U937 cell line U1/HIV-1 (U1) chromatin induced by TPA treatment orexogenous viral Tat expression indicated that activated provirus exists in a transcription-dependent loop conformation that juxtaposes the 5 long terminal repeat (LTR) promoter and3LTR terminator regions. Based on ChIP analysis, RNA polymerase II (Pol II) and factorsinvolved in transcriptional initiation and elongation (Cdk9 and USF) were detected at bothends of the HIV-1 provirus, supporting the existence of LTR-LTR interaction upon activationof proviral reservoirs. To determine whether proviral loop formation occurs before or afterCdk9-mediated Pol II CTD phosphorylation, we are currently performing 3C and ChIPanalysis on chromatin treated with flavopiridol, a specific inhibitor of Cdk9 kinase activity.We propose that when latent integrated HIV-1 proviral DNA is transcriptionally activated inreponse to cellular or Tat-mediated stimulation, a structural formation is created where theflanking LTRs reside in close spatial proximity. This permits “cross-talk” between initiationand termination factors and so may allow efficient recycling of transcriptional machinery.This is the first study to identify the spatial arrangement of active integrated HIV-1 provirusand may provide valuable insight into the physical properties of HIV-1 proviral reservoirsupon transcriptional activation.

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David Picketts Abstract P89SNF2L-mediated control of cell number in the developingbrain

Darren J. Yip1,3, Stephen Rennick1,3, Adriana de Maria1, Josée Coulombe1,Michael Rudnicki1,2 and David J. Picketts1,3

1Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa,ON, 2Department of Cellular and Molecular Medicine and 3Department of Biochemistry,Microbiology and Immunology, University of Ottawa, Ottawa, ON

Epigenetic modification of the genome is becoming recognized as a major point ofregulation governing many developmental processes. Certainly, defective epigeneticregulation is already implicated in a wide range of genetic disorders including mentalretardation, autoimmune disorders, and cancer. We have shown that the ISWI family ofchromatin remodelling proteins are abundantly expressed during mammalian braindevelopment and thus, may be key participants in establishing chromatin domains that arecharacteristic of neurons. Here we used a conditional gene-targeting approach to inactivatethe murine Snf2l gene in order to assess its role in neurodevelopment. Heterozygousfemales (Snf2lf/x) were crossed to mice expressing Cre-recombinase under the control ofthe GATA-1 promoter. SNF2L-null male mice were viable and born at classic Mendelianratios. These mice displayed no overt developmental or behavioral abnormalities; however,the loss of Snf2l resulted in a 2-fold increase in the brain weight to body weight ratio. Thiswas accompanied by a concomitant increase in cell number in the hippocampus andcerebral cortex ranging from 0.5-2-fold in the six distinct cortical layers and a 2-fold increasein all hippocampal strata. Using a combination of in situ BrdU labeling experiments andprimary neuroprogenitor cultures we demonstrate that the increased cell number resultsfrom a delay in terminal differentiation. Indeed, neuroprogenitor cultures isolated fromknockout animals continue to incorporate BrdU twice as long as wild type cells wheninduced to differentiate. As a result, fewer cells stain positive for neuronal or astrocyticmarkers at earlier times (4 days) but are equivalent by the end of the 7-day differentiationtime course. Taken together, our results suggest that Snf2l has an important role inregulating the switch from proliferation to differentiation as a mechanism to control cellnumber and brain size during neuronal development. Moreover, we propose that Snf2lregulation of brain size occurs through direct effects on genes involved in cell cycle exitand/or terminal differentiation.

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Romina Ponzielli Abstract P90Optimization of experimental design parameters of ChIP-on-chip studies

Romina Ponzielli1 Paul C. Boutros1,2,3 , Sigal Katz1 , Igor Jurisica1,3,4 andLinda Z. Penn1,2

1Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University HealthNetwork, Toronto, Canada M5G 2M9, 2Department of Medical Biophysics, University ofToronto, Canada, 3Division of Signaling Biology, Ontario Cancer Institute, University HealthNetwork, Toronto, Canada M5G 2M9, 4Department of Computer Science, University ofToronto, Canada

Chromatin immunoprecipitation (ChIP) is an antibody-based technique for determiningtranscription-factor binding in vivo. ChIP-chip technology combines the sensitivity andspecificity of ChIP with high-throughput analysis, by exploiting microarray platforms spottedwith promoter region DNA. In yeast, this technique has been used to map transcription-regulatory networks; in mammals, ChIP-on-Chip has determined transcription-factor bindingin normal and cancerous cells and tissues. While ChIP-on-Chip studies are yielding usefulresults, their bioinformatic analysis is not yet fully realized. In this study we characterizeboth the statistical and experimental-design features of ChIP-on-Chip.To profile the signal-to-noise characteristics of ChIP-on-Chip data we performed a series ofvalidation studies. First, we compared the performance of different amplification methods.Second, we determined optimal sample-to-array allocation. Third, we profiled the effect ofdye-bias. Fourth, we evaluated effects of array batch variability. Finally, we determined theimportance of antibody purity for successful ChIP-on-Chip studies. These studiesencompass over 100 arrays, the data from which was exploited in a large-scale empiricalstudy of statistical pre-processing methods. We assessed 84 distinct analysis methods forsensitivity, stability, and selectivity. Through these analyses we have optimized the majordesign parameters of ChIP-on-Chip studies. Our rigorous characterization of ChIP-on-Chipdata is a key step towards exploiting this important technology for the rapid elucidation ofregulatory networks.

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Ryan Raisner Abstract P91Single nucleosome resolution mapping of the histone variantH2A.Z in a developing organism

Ryan M Raisner and Hiten D Madhani

UCSF Department of Biochemistry and Biophysics, U.S.A.

The conserved histone variant H2A.Z is an essential protein in higher organisms that isrequired early in development. It is poorly understood where H2A.Z is localized inmetazoans, and furthermore what it’s essential biological function is. It has been shown byseveral groups that in the budding yeast Saccharomyces cerevisiae, H2A.Z is presentthroughout the genome, and more specifically it marks the promoter regions of the majorityof genes, regardless of their transcription rate. More recently, there has been evidence thatH2A.Z can selectively mark active genes in vertebrates. We have chosen the modelorganism Danio rerio (zebrafish) to study the distribution of H2A.Z using chromatinimmunoprecipitation along with high resolution tiling microarrays at a range of genes,including the developmentally important Hox gene clusters at several stages of thedeveloping animal. In addition to H2A.Z, we have also profiled the chromatin modificationsH3 tri-methyl lysine 27 and tri-methyl lysine 4 at the same regions at single nucleosomeresolution. These modifications have been shown recently to be present predominantly attranscription factor promoters in pre-differentiated cells. Accordingly, their coincidence withH2A.Z, along with the dynamics of all three chromatin marks at the Hox gene clustersthroughout development is of great general interest. Our localization studies of H2A.Z in thedevelopmentally tractable vertebrate system of zebrafish will allow us to better understandthe nature of the activity of H2A.Z in the context of a complex developing organism.

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Rama Natarajan Abstract P92Genome-wide analysis of histone lysine methylationvariations caused by diabetic conditions in human monocytes

Feng Miao1, Xiwei Wu2, Lingxiao Zhang1, Yate-Ching Yuan2, Arthur D.Riggs3 and Rama Natarajan1

Departments of Diabetes1, Biomedical Informatics2 and Biology3, Beckman ResearchInstitute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, U.S.A.

Histone modifications in chromatin, particularly histone lysine methylations, play keyepigenetic roles in gene expression and constitute a new layer of transcription regulation.Aberrant methylation patterns that change chromatin structure can promote dysregulatedgene expression associated with disease progression. Current approaches for acquiringgenome-wide information of histone modifications involve the use of chromatin-immunoprecipitation linked to DNA arrays (ChIP–arrays). This method generates data setsof chromatin status and provides snapshot views of the cell at the layer of histonemethylation.Diabetic conditions such as high glucose (HG) can alter key pathologic pathways andgenes. However, their impact on cellular histone lysine methylation is not known. Wehypothesized that chronic HG can induce aberrant changes in histone H3 lysine-4 andlysine-9 dimethylation (H3K4me2 and H3K9me2), key histone marks normally associatedwith active and repressed genes respectively. We used antibodies to H3K4me2 andH3K9me2 in ChIP-arrays to compare their profiles and variations in human THP-1monocytes cultured in normal glucose (NG) and HG separately. We used human 12K cDNAand 12K CpG arrays representing coding and promoter regions respectively. After statisticalanalyses of the ChIP-microarray data, we identified key candidate genes relevant todiabetes that displayed changes in H3K4me2 and H3K9me2 in HG relative to NG and alsovalidated them with follow-up conventional ChIPs. Relevance to diabetes was furtherdemonstrated by examining these modifications by ChIPs in peripheral blood monocytesisolated from patients with type 1 diabetes relative to normal controls. In addition, regularmRNA profiling with the cDNA arrays revealed both anticipated and unanticipatedcorrelations between mRNA expression, H3K4me2 and H3K9me2 levels. These resultsshowing HG-and diabetes-induced variations in histone methylation genome-wide suggestthat the diseased cells may have distinct epigenomes and also provide new insights intodiabetic changes in the context of histone methylation. This may lead to an epigeneticmemory of transcription history responsible for the “metabolic memory” and sustainedclinical complications of diabetes.

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Edward Ramos Abstract P93Global characterization and function of Gypsy-likeendogenous insulators in Drosophila melanogaster

Edward Ramos and Victor Corces

Johns Hopkins Univ, Dept of Biol Mudd Hall, 3400 N Charles St, Baltimore, MD 21218,U.S.A.

Higher-order chromatin organization is essential for the proper expression of eukaryoticgenes. The chromatin domains established through this organization are thought to regulategene expression by controlling intra-chromosomal domain communication. The gypsychromatin insulator proteins of Drosophila melanogaster have been implicated in theestablishment of these domains. However, the mechanism governing the genomicorganization and maintenance of these elements is not well understood. To betterunderstand the role of insulators in chromatin organization, we investigated the function ofthe insulator protein Su(Hw) at the global level. To this end we used a combination oftraditional molecular, biochemical and genetic approaches along with novel computationaltools for our studies. We have thoroughly characterized a number of endogenous Su(Hw)binding sites and determined, through gel shift binding assays, direct interaction betweenpredicted Su(Hw) binding sites and Su(Hw) insulator protein. In addition, in vivo experimentsdemonstrated that these predicted endogenous Su(Hw) binding sites co-localize withSu(Hw) protein on polytene chromosomes. Finally and most importantly, these endogenousSu(Hw) binding sites function as bona fide insulators as they can prevent communicationbetween enhancers and promoters as demonstrated in an enhancer blocking assay. Usingcomputational analysis we are in the process of mapping these Su(Hw) binding site to theentire Drosophila genome to determine the global function of the Su(Hw) protein during flydevelopment and everyday function. It is our hope to find a functional correlation betweenour endogenous insulator binding sites and global genomic regulation

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William Renthal Abstract P94Class II histone deacetylases regulate the behavioraladaptations to chronic cocaine and stress

William Renthal1, Arvind Kumar1, Vaishnav Krishnan1, Scott J. Russo1,David E. Theobald1, Kwang-Ho Choi1, Nadia Tsankova1, Rachel Neve2,Eric N. Olson3 and Eric J. Nestler1

1Department of Psychiatry, UT Southwestern Medical Center, 2Department of Psychiatry,McLean Hospital, Harvard Medical Center, 3Department of Molecular Biology, UTSouthwestern Medical Center, U.S.A.

Addiction and depression are chronic psychiatric disorders in which long-lasting changes ingene expression are thought to contribute to the behavioral pathologies. Previous studiesfrom our lab have identified alterations in histone acetylation in rodent models of addictionand depression that may contribute to the longevity of these disorders. Sinceneurotransmission is highly dependent on calcium signaling, we focused on the calcium-regulated class II histone decacetylases (HDACs) as potential mediators of the addictionand depression-induced changes in chromatin structure. Calcium influx results in the rapidphosphorylation and nuclear exportation of these HDACs, which in turn leads to asubsequent increase of histone acetylation on target genes. Mice lacking the class IIHDACs, HDAC5 or HDAC9, are completely normal in a variety of behavioral tests fromlearning and memory to acute cocaine reward. However, after chronic cocaine treatmentsor chronic stress, they demonstrate increased sensitivity to both stimuli. These mice seemto hyper-adapt, suggesting that proper balance of histone acetylation is important in thebehavioral adaptations to chronic but not acute stimuli. We have also found that cocaineand stress regulate HDAC5 phosphorylation in wild type mice, which corresponds withchanges in histone acetylation of HDAC5 target genes that were identified by geneexpression microarrays and ChIP on chip technology. We are currently investigating therole of these HDAC5 target genes individually using viral-mediated gene transfer intospecific nuclei in the brain. These studies together with analogous findings in models ofchronic cardiac stress, implicate class II HDACs in the molecular machinery needed foradapting to a variety of chronic environmental stimuli.

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Karsten Rippe Abstract P95Activities of histone chaperone NAP1: Association states andinteractions with histones, nucleosome assembly and effecton the chromatin fiber conformation

Karsten Rippe, Jacek Mazurkiewicz, Felix Kepert and Katalin Fejes Toth

Kirchhoff-Institut fur Physik, Molecular Biophysics Group, Ruprecht-Karls-UniversitatHeidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany

The nucleosome assembly protein 1 (NAP1) is a histone chaperone that functions as acarrier of histones during nuclear import, nucleosome assembly and chromatin remodeling.We have examined (i) the association states of NAP1 alone and in complexes with histones[1], (ii) the process of mononucleosome assembly mediated by NAP1 on DNA fragments of146 and 207 bp length containing a 5 S rDNA nucleosome positioning sequence [2], and(iii) how the interaction of NAP1 with core and linker histones affects the chromatin fiberorganization [3]. Taken together these results provide complementary insight into themechanisms, by which NAP1 exerts its different biological activites.

Refs:1. Fejes Toth, K., Mazurkiewicz, J. & Rippe, K. (2005). J. Biol. Chem. 280, 15690-15699.2. Mazurkiewicz, J., Kepert, J.F. & Rippe, K. (2006). J. Biol. Chem. 281, 16462-16472.3. Kepert, J.F., Mazurkiewicz, J., Heuvelman, G., Fejes Toth, K. & Rippe, K. (2005). J. Biol.Chem. 280, 34063-34072.

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Charles Roberts Abstract P96The Swi/Snf chromatin remodeling complex regulateslineage specific transcription programs during developmentand impairment of this activity causes cancer

Xi Wang, Miriam B. Werneck, Courtney G. Sansam, Michael S. Isakoffand Julia A. Evans

Harvey Cantor and Charles W. M. Roberts. Dana-Farber Cancer Inst., Boston, MA, U.S.A.

Chromatin remodeling complexes control nucleosome positioning and are dynamicregulators of transcription. While the biochemical role of these complexes in nucleosomemobilization is beginning to be elucidated, little understanding exists with respect to the roleof the complexes in coordinated control of gene expression programs such as thoserequired for lineage specific development. Snf5/Ini1/Baf47/Smarcb1 is a core member of theSwi/Snf chromatin remodeling complex and is a potent tumor suppressor that is inactivatedin aggressive childhood cancers. As many transcription factors involved in oncogenictransformation are master regulators of lineage specific development, we hypothesized thatthe Swi/Snf complex, which also regulates transcription, may serve a similar role and thatthis activity may underlie its role in tumor suppression. We therefore sought to identify thedevelopmental function of Snf5 and to elucidate the mechanistic basis of its tumorsuppressor activity. Since the progressive stages of T cell development are wellcharacterized and because Snf5 loss gives rise to T cell lymphomas, we chose toinvestigate the role of Snf5 in T cell development. We found that deletion of Snf5 in miceleads to aberrant gene expression in T cells, abnormal developmental progression withimbalanced CD4:CD8 bifurcation and culminates in a specific developmental block. Thisdysfunction and imbalance is coupled with the extremely rapid formation of mature CD8+ Tcell lymphomas in 100% of mice. This developmental role is highly specific for alpha-beta Tcells as we show that Snf5 is largely dispensable for T cells of the gamma-delta lineage andthat it possesses no tumor suppressor activity within the gamma-delta or B cell lineages.Lastly, we show that the tumor suppressor activity is likely derived from the role of Snf5 inrestricting cell cycle progression/proliferation and promoting differentiation. Loss of Snf5leads to aberrant stimulation of the cell cycle by causing down-regulation of p16Ink4a andactivation of E2F targets which in turn directly drive replication. These changes promotecancer formation. Collectively, our data demonstrate a role for the Swi/Snf chromatinremodeling complex in controlling lineage specific developmental programs and provideinsight into how loss of this control leads to oncogenic transformation.

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Paul Sadowski Abstract P97Post-translational modification of the insulator protein, CTCF

Melissa MacPherson, Linda Beatty, Wenjing Zhou and Paul Sadowski

Department of Medical Genetics and Microbiology, University of Toronto, TorontoM5S 1A8 Canada

The CTCF protein is a highly conserved zinc finger protein that has been implicated in manyaspects of gene regulation and nuclear organization. It acts as a repressor of some genessuch as the c-myc gene but it activates others such as the ß-amyloid precursor proteingene. It also plays a key role in regulating genomic imprinting of the IGF2 and H19 genesby binding to a differentially methylated insulator sequence that lies between the two genes.The CTCF protein may be over expressed or mutated in some cancers. It is post-translationally modified by poly(ADP) ribosylation and is phosphorylated by casein kinase II.We now report that CTCF is also post-translationally modified by the ubiquitin-like proteins,SUMO. We have co-transfected HEK293 cells with plasmids encoding tagged versions ofCTCF and SUMOs 1, 2 or 3 followed by immunoprecipitation and western blotting to revealthat CTCF is SUMOylated by SUMOs 1, 2 and 3. The reaction is dependent upon the C-terminal diglycine amino acids of SUMOs 1 and 2 and is sensitive to the SUMO proteasesSENP1 and SENP5 (vectors supplied by Dr. E. Yeh). The SUMOylation reaction isinsensitive to 3-aminobenzamide, an inhibitor of poly (ADP) ribose polymerase. Thus far, wehave identified one strong SUMO acceptor site in the C-terminus of CTCF. SUMOylation ofCTCF also occurs efficiently in vitro and SUMOylation does not affect CTCF’s ability to bindto DNA that contains CTCF-binding consensus sequences from the H19 insulator.We are studying the interrelationship of SUMOylation of CTCF with other post-translationalmodifications of the protein and its influence upon the multitude of transcriptional activitiesof this protein.(Supported by the Canadian Institutes of Health Research)

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Teresa Sanchez Alcaraz Abstract P98Role of USP7 and GMP synthetase in deubiquitination ofhuman histone H2B

Teresa Sanchez Alcaraz1, Melissa Holowaty1, Yi Sheng2, CherylArrowsmith2 and Lori Frappier1

1Department of Medical Genetics and Microbiology and 2Banting and Best Department ofMedical Research, University of Toronto, Toronto, Canada

One of the post-translational modifications that histones can undergo is monoubiquitination.This modification occurs predominantly on H2A and H2B but is poorly understood in termsof its effect on chromatin structure and function, as well as the enzymes controlling theubiquitination/deubiquitination process. In mammalian cells, histone H2B has been shownto be ubiquitinated by Mdm2 and RFN20/40 but the ubiquitin-specific protease (USP) thatreverses this modification is not known. We have been studying the structure and function ofUSP7 in human cells (also called HAUSP), which is the target of herpes simplex andEpstein-Barr virus proteins (ICP0 and EBNA1) and plays roles in p53 regulation (2005 Mol.Cell 18, 25; 2006 Nature Struc. Mol. Biol. 13, 285). To more completely determine howUSP7 affects cellular processes, we investigated protein interactions of USP7 using anaffinity column approach. We found that the most predominant interaction of USP7 is withhuman GMP synthetase. Interestingly, recent studies in Drosophila indicate that H2B isdeubiquitinated by USP7 in complex with GMP synthetase (2005 Mol Cell 17, 695). Wefound that the USP7-GMP synthetase interaction occurs through the USP7 N-terminaldomain, which is also responsible for binding p53, Mdm2 and EBNA1. We have shown thatpurified USP7 has some capacity to cleave ubiquitin from monoubiquitinated H2B isolatedfrom human cells, but that this activity is greatly stimulated by purified GMP synthetase.This effect was specific for H2B, as deubiquitination of polyubiquitinated p53 by USP7 wasnot affected by GMP synthetase. Consistent with these observation, cellular levels ofubiquitinated H2B were decreased when USP7 was silenced in Hela cells by a hairpin RNA.The results suggest that a complex of USP7 and GMP synthetase is responsible fordeubiquitination of H2B in higher eukaryotes.

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Annette Scharf Abstract P99Dynamics of histone modifications during chromatinassembly

Annette ND Scharf, Ana Villar-Garea and Axel Imhof

Adolf Butenandt Institute, Schillerstrasse 44, 80336 Munich, Germany

Histone tails undergo a variety of posttranslational modifications that change theirinteraction with DNA and nuclear proteins. Combinations of modifications are thought toserve as a ‘histone code’. Changes in combinatorial histone modifications mark uniquedownstream events such as gene expression and DNA repair. Moreover histonemodifications are thought to be transmitted through mitosis and therefore serve asmediators of cellular memory. However the way histone modifications are passed on fromone cell generation to the other remains elusive. Using well-established MALDI-TOF andESI MS/MS techniques our goal is to describe the changes in histone modifications duringchromatin assembly. Therefore we use a Drosophila melanogaster assembly extract derivedfrom preblastoderm embryos to assemble chromatin in vitro. Here, we find that there is agradual deacetylation of H4K5 and H4K12 during the assembly, and that the deacetylationis dependent on ATP. We also confirm that the deacetylation is sensitive to HDAC inhibitors.We are currently investigating these findings in vivo by means of stable isotope labeling withamino acids in cell culture (SILAC).

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Stefan Schoeftner Abstract P100Screening for miRNAs regulating mammalian telomeres

Stefan Schoeftner1, Susana Gonzalez1, Manuel Serrano1, Gregory J.Hannon2 and Maria A. Blasco1

1Spanish National Cancer Center (CNIO), 28029 Madrid, Spain; 2Cold Spring HarborLaboratory, Cold Spring Harbor, New York 11724, U.S.A.

Mammalian telomeres are specialized chromatin structures at the ends of chromosomesthat are essential for genomic stability. Telomeres shorten witch age due to the so called“end-replication problem”, a process underlying ageing.Telomere length is regulated by proteins binding directly to telomeric TTAGGG tandemrepeats but also by chromatin modifying enzymatic activities such as histone lysinemethyltransferases (HMTases) and DNA methyltransferases (DNMTs).Recently, microRNAs (miRNAs) were identified to play a central role in regulating animaldevelopment and physiology. miRNAs are ~21-26 nucleotide RNAs that regulate their targetRNA by triggering endonuclease driven cleavage or translational repression. MammalianmiRNAs are generated from PolII transcribed precursor RNAs and processed into an activeform by cleavage by the RNase III enzymes Drosha and Dicer. Importantly, mouse ES cellslacking Dicer show dramatically elongated telomeres with altered chromatin structure*. Thisfinding suggests that miRNAs could be directly or indirectly involved in the regulation oftelomere function and contribute to telomere associated diseases.In order to identify miRNAs involved in the regulation of telomeres we are taking advantageof a miRNA library comprising a set of 150 predicted miRNAs.The miRNA containing vectors were transiently transfected in to a Hela cell line carrying aluciferase reporter gene in close proximity to a telomere. miRNA induced changes intelomeric or subtelomeric chromatin result in altered expression of the reporter gene, aphenomenon known as “telomere position effect” (TPE). Additionally, the miRNA library wastransfected into a standard Hela cell line to study changes in telomere length and globalchromatin structure using high throughput quantitative fluorescence hybridisation (Q-FISH)and quantitative immunofluorescence (Q-IF).The poster outlines the primary screening as well functional validation of candidate miRNAsregulating telomere length and telomeric chromatin.

*Benetti et al., unpublished data

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Gunnar Schotta Abstract P101A genome-wide transition to H4K20 mono-methylationimpairs stress-induced and programd DNA damage responsein the mouse

Gunnar Schotta, Roopsha Sengupta, Stefan Kubicek, Stephen Malin,Michaela Pagani, Monika Kauer, Alexsandra Espejo, Mark Bedford,Meinrad Busslinger and Thomas Jenuwein

Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, A-1030 Vienna,Austria; University of Texas, M.D. Anderson Cancer Center, Science Park ResearchDivision, PO box 389, Smithville, Texas 79857, U.S.A.

H4K20 methylation is a broad epigenetic modification that has been linked with genesilencing, heterochromatin formation and response of the chromatin template toenvironmental signals. To analyze its function during mammalian development, we havedisrupted the two Suv4-20h HMTases in the mouse. Whereas Suv4-20h1 null mutantsdisplay perinatal lethality, Suv4-20h2 deficient mice develop normally. Using conditionalalleles, we generated Suv4-20h double null (dn) mice, in which nearly all H4K20me3 andH4K20me2 states are lost, resulting in a genome-wide transition to H4K20me1 chromatin.Suv4-20h dn mouse embryonic fibroblasts (MEFs) display impaired proliferation, havereduced S-phase ratios and enter crisis at early passage numbers. The DNA damagecheckpoint protein 53bp1 selectively associates with H4K20me2 nucleosomes, andrecruitment of 53bp1 to DSBs is significantly impaired in Suv4-20h dn cells. Importantly,Suv4-20h dn B-cells are also defective in class switch recombination, which reflects adevelopmentally programd pathway for DSB-mediated antibody isotype diversification. Thus,an H4K20me1 chromatin is insufficient to ensure mammalian genome function andH4K20me2 is a major signal in processing stress-induced and programd DNA damageresponse in the mouse.

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David Schrump Abstract P102Brother of the Regulator of Imprinted Sites (BORIS) recruitsSp1 to modulate NY-ESO-1 expression in lung cancer cells

Yang Kang, Julie A. Hong, G. Aaron Chen, Dao M. Nguyen and David S.Schrump

Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National CancerInstitute, Bethesda, MD, U.S.A.

Epigenetic alterations during malignant transformation facilitate de-repression of a variety ofgerm cell-restricted genes, such as NY-ESO-1, which encodes a cytoplasmic protein that isimmunogenic in cancer patients. Recently we reported that the paralogous zinc fingerproteins- CTCF and BORIS, directly contribute to transcriptional regulation of NY-ESO-1 inlung cancer cells. To further examine mechanisms that mediate NY-ESO-1 expression, weperformed software-guided analysis of the NY-ESO-1 promoter region, which revealedseveral potential Sp1 binding motifs. Promoter-reporter assays demonstrated that deletions,which sequentially eliminated the putative BORIS/CTCF recognition sequence and aprototypic Sp1 binding site markedly decreased NY-ESO-1 promoter activity. Transienttransfection experiments using promoter-reporter constructs, electromobility shift assays,and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activitycoincided with increased occupancy of the proximal Sp1 binding site in lung cancer cells.Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to thismotif in vitro. siRNA-mediated inhibition of Sp1 expression coincided with markedlydiminished NY-ESO-1 promoter activity in lung cancer cells. In contrast, abrogation of CTCFexpression resulted in pronounced augmentation of NY-ESO-1 promoter activity. Co-immunoprecipitation experiments indicated that Sp1 physically interacts with BORIS but notwith CTCF in vivo. Collectively, these findings suggest that NY-ESO-1 expression isgoverned by distinct transcriptional complexes during pulmonary carcinogenesis, and thatBORIS recruits Sp1 to augment NY-ESO-1 expression in lung cancer cells.

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Bonnie Scott Abstract P103Evolution of centromere-binding proteins and theirinteractions with centromere DNA in Arabidopsis

Bonnie Scott, Song Luo, Sarah Hall and Daphne Preuss

Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology,GCIS W519, University of Chicago, 929 E. 57th Street, Chicago, IL 60637, U.S.A.

During cell division, proper chromosome segregation involves assembling a kinetochoreprotein complex at the centromere region of every chromosome. A fundamental yetunresolved question is how inner kinetochore proteins initially choose the site forkinetochore assembly and subsequently maintain its identity as a centromere throughoutthe cell cycle. The challenge in understanding these phenomena is that inner kinetochoreproteins, namely centromere-binding proteins (CENP) A and C, share high sequencesimilarity between plants, yeast, and animals, yet the centromere DNA to which theyassociate lacks a conserved sequence and is instead composed of repetitive DNA,including rapidly evolving satellite DNA. These observations have led to the hypothesis thatCENPs evolve adaptively across species to maintain protein-DNA interactions at thecentromere for chromosome inheritance. The goal of this study is to identify structuralfeatures within plant centromere-binding proteins that are required for species-specificcentromere function in vivo. To accomplish this, we have first cloned CENP-A and CENP-Csequences from increasingly diverged Arabidopsis species and used phylogenetic tools tounderstand their evolutionary history and identify putative regions important for species-specificity. Second, we are expressing these CENP sequences into correspondingArabidopsis thaliana mutants to define the degree of natural variation able to rescue CENPfunction at the centromere.

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David Shechter Abstract P104Histone H2A arginine3 is mono- and symmetrically-dimethylated by a complex of PRMT5 and the WD-repeatprotein MEP50 in Xenopus laevis eggs

David Shechter1, Raghu Chitta2, Eileen Woo3, Brian Chait3, JeffreyShabanowitz2, Donald F. Hunt2 and C. David Allis1

1The Laboratory of Chromatin Biology, Rockefeller University, New York, NY 10021;2Chemistry Department, University of Virginia, Charlottesville, VA 22904; 3The Laboratory ofMass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY10021, U.S.A.

A wealth of emerging literature demonstrate that post-translational modification of histones, theprotein components of nucleosomes which are the fundamental repeating unit of chromatin, serveto regulate a wide variety of DNA-templated processes, notably gene regulation. Methylation ofarginines in histones has been observed in many contexts, including gene activation andrepression [1]. Histone arginine methylation has been correlated with events during development,especially in germ cells. Here we report the identification of the predominant histonemethyltransferase in extracts of the large eggs of Xenopus laevis. The activity is composed of, atminimum, a complex of the methyltransferase PRMT5 (also known as Hsl7 in Xenopus andyeast) and the WD-Repeat Protein MEP50. It specifically catalyzes mono- and symmetric-dimethylation on free histone H2A on arginine 3, as determined by mass spectrometry and byimmunoblotting with H2A/H4R3me-specific antibodies. The complex methylates H2A and H4 innucleosomes at a much lower activity. The methylation activity towards H2A is inhibited by H2ASerine-1 phosphorylation, although we did observe H2AS1phos and H2AR3Me on the sameprotein molecule by mass spectrometry. AMI-1, a general small molecule inhibitor of PRMTs,inhibits this complex at approximately 100µM. This activity and H2AR3Me2S modification isabundant in the egg and disappears rapidly during development. In conclusion, H2AR3methylation is a major histone modification in the early development of Xenopus laevis.Experiments are in progress to gain insight into the biological function(s) of PRMT5/MEP50complex in the context of early development. We also are exploring whether MEP50 serves topresent H2A to PRMT5, analogous to the function for the WD-Repeat Protein WDR5 in MLL(mixed lineage leukemia) complex isolated from human cells as recently described by ourlaboratory [2, 3].

Refs1. Bedford, M.T. and S. Richard, Arginine methylation an emerging regulator of protein function.Mol Cell, 2005. 18(3): p. 263-72.2. Dou, Y., et al., Regulation of MLL1 H3K4 methyltransferase activity by its core components. NatStruct Mol Biol, 2006. 13(8): p. 713-9.3. Ruthenburg, A.J., et al., Histone H3 recognition and presentation by the WDR5 module of theMLL1 complex. Nat Struct Mol Biol, 2006. 13(8): p. 704-12.

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Yoichi Shinkai Abstract P105H3K9 methylation and germ cell development

Makoto Tachibana1, Masami Nozaki2, Naoki Takeda3 and Yoichi Shinkai1

1Institute for Virus Research, Kyoto University, 2Research Institute for Microbial Disease, OsakaUniversity, 3Center for Animal Resources and Development, Kumamoto University, Japan

It is known that epigenetic cellular memories are dynamically reprogramd during germ celldevelopment. G9a is one of major histone H3 lysine 9 (H3K9) methyltransferases ateuchromatin. To elucidate how G9a and G9a-mediated H3K9 methylation are crucial forgerm cell development, we established germ-lineage specific G9a deficient mice. In theabsence of G9a, male and most female mice are sterile. Even TNAP (tissue non-specificalkaline phosphatase) promoter-driven Cre enzyme supposedly inactivates a G9a gene (andG9a-mediated H3K9 methylation) before the migration stage into testis, spermatogenesis inG9a-deficient mice until a gonia type stage seems to be intact. However, spermatocyteslater a pachytene stage are missing. Furthermore, abnormal meiotic-prophase progressionand synaptonemal complex formation are frequently observed in G9a-deficientspermatocytes. Oogenesis is also impaired around the pachytene stage. DNA microarrayanalysis described that multiple genes are activated or up-regulated in G9a-deficienttesticular cells. These results emphasize a crucial function for G9a (and G9a-mediatedH3K9 methylation) in controlling germ cell development and meiotic-prophase progression.

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Krishna Sinha Abstract P106Inhibition of the transcriptional activity of osterix byinteractions with NO66, a jumonji family chromatin protein

Krishna M Sinha, Xin Zhou, Chi Zhang, Lingna Zhang and Benoit deCrombrugghe

Department of Molecular Genetics, UT M. D. Anderson Cancer Center, Houston,TX 77030, U.S.A.

Osterix (Osx) is a critical osteoblast-specific transcription factor required for bone formation.Genetic inactivation of Osx in mice leads to complete arrest of osteoblast differentiation,although chondrocyte differentiation and cartilage formation are normal. However, themechanism of Osx function during osteoblast differentiation is not well understood.In the present study, the Jumonji (JmjC) domain containing protein, NO66, was identified asan Osx-interacting protein using an Osx expressing stable osteoblast cell line by tandemimmunoaffinity chromatography and mass spectrometry. Co-immunoprecipitation and GST-pulldown assays showed that NO66 physically interacts with Osx. NO66 interacts throughits JmjC domain with the transcription activation domain of Osx. In situ RNA hybridizationexperiments revealed that NO66 and Osx are co-expressed in skeletal elements of hindlimbs and forelimbs, in vertebrae, ribs, and craniofacial bones during mouse embryonicdevelopment. In DNA transfection assays, Osx strongly stimulates the activity of a 1kbosteocalcin promoter and that of an osteoblast specific 2.3-kb Col1a1 promoter. Co-transfection of NO66 results in a strong inhibition of the Osx-dependent activity of thesepromoters, whereas the activity of several other promoters was not affected by NO66. Inaddition to this, transfection of NO66 in osteoblasts also inhibits the activity of theendogenous osteocalcin gene. Furthermore, knockdown of Obelix expression in osteoblastsby specific siRNAs results in increased expression of several osteoblast-specific markergenes including Col1a1, Osteocalcin and Bone Sialoprotein (BSP) without apparentchanges in the level of Osx expression.Overall our data suggest the hypothesis that NO66 is a negative regulator of Osx and,hence, of osteoblast differentiation or function. The presence of a JmjC domain in NO66raises the intriguing possibility that inhibitory effect of NO66 is mediated by demethylation ofspecific lysine residues of histones within the chromatin environment of osteoblasts.

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Karen Smith Abstract P107Identification and characterization of novel HDAC-associatedproteins that regulate cancer cell growth

Karen T. Smith, Skylar A. Martin-Brown, Laurence Florens, Michael P.Washburn and Jerry L. Workman

The Stowers Institute for Medical Research, Kansas City, MO 64110 U.S.A.

Histone deacetylase (HDAC) inhibitors are one class of chemotherapeutic drugs. However,these inhibitors exhibit undesirable side effects in cancer patients due to their inability todiscriminate among the several structurally similar HDACs in humans. HDACs reside inmulti-subunit protein complexes and some of these HDAC-associated proteins are neededfor full activity of the HDACs themselves. We propose that a single HDAC can be inhibitedin the cell by targeting HDAC-associated proteins rather than the HDACs themselves. Wehave taken a proteomics approach to identify proteins differentially associated with humanHDAC1 and HDAC3 in 293T cells. Preliminary experiments have identified several knownmembers of HDAC-containing complexes and many novel HDAC-interacting proteins.Importantly, many of these proteins differentially associate with HDAC1 or HDAC3. Severalof these newly identified HDAC1 and HDAC3-interacting proteins have a connection tochromatin or cell growth, while others have not yet been described. We are currentlyconfirming these interactions in 293T cells and testing the conservation of theseinteractions in cancer cell lines. We then will test if these HDAC-interacting proteins areimportant for their associated HDAC’s activity. Proteins that have a significant effect onHDAC1 or HDAC3 activity will then be abrogated in cancer cell lines to determine if loss ofthese HDAC-associated proteins can inhibit cancer cell growth.

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Matthew Smith Abstract P108Chromatin - mediated silencing of immune response genes

Matthew Smith, Jian Wu and Kenneth Wright

University of South Florida College of Medicine, Department of Molecular Medicine andMoffitt Cancer Center Tampa, FL, U.S.A.

The transcriptional repressor PRDI-BF1 (Blimp-1) is required for terminal differentiation ofplasma cells, T-cell homeostasis and silencing of interferon beta expression upon viralinduction. The repressive functions of PRDI-BF1 are mediated through interactions withadditional co-factors, such as Groucho, HDAC1/2 and G9a. Here, we demonstrate a role forPRDI-BF1 in the maturation of dendritic cells (DCs). Considerable phenotypic differencesexist between immature and mature DC populations. Immature DCs function in immunesurveillance via antigen uptake and processing, while mature DCs present antigen in thecontext of MHC Class II molecules to immune effector molecules. This phenotypic transitioninvolves an orchestrated change in gene expression profiles, exemplified by the down-regulation of CIITA and MRC1 upon maturation. CIITA, encoded by MHC2TA, is the masterregulator of MHC Class II transcription which is known to be silenced upon maturation.MRC1 (CD206) recognizes extracellular pathogens via oligosaccharide domains and iscapable of activating the innate immune response. PRDI-BF1 is induced upon DCmaturation (antigen encounter) and inversely correlates with expression of MHC2TA andMRC1 mRNA. Using in vivo genomic footprinting and chromatin immunoprecipitationexperiments, we show that transcriptional co-activators are displaced by PRDI-BF1 at thepromoter regions of MHC2TA and MRC1. Upon maturation, these regions demonstratehallmarks of silent chromatin, such as decreased acetylation of histones H3 and H4,decreased methylation of histone H3 lysine 4 and increased di-methylation of histone H3lysine 9. Furthermore, this silencing is mediated by the G9a histone methyltransferase andthe heterochromatin protein, HP1 gamma. These results provide novel insight into themolecular mechanisms underlying the maturation process in DCs.

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Hae-Ryong Song Abstract P109Coordination of transcriptional regulation and chromatinmodification of Arabidopsis circadian clock genes

Hae-Ryong Song1,3, Ju-Hee Jeong1,3, Bosl Noh2,3 and Yoo-Sun Noh1,3

1Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea.,2Environmental Biotechnology National Core Research Center, Gyeongsang NationalUniversity, Jinju 660-701, Korea. 3Global Research Laboratory for Flowering at SNU andUW, Seoul National University, Seoul 151-742, Korea

Circadian clock genes are regulated through a transcriptional-translational feedback loop. InArabidopsis, LHY and CCA1 transcripts are highly expressed in the early morning.Translated LHY and CCA1 proteins repress the expression of TOC1 transcript which peaksin the evening. TOC1 protein elevates the expression of LHY and CCA1 mRNAs, forming anegative feedback loop that is believed to constitute the oscillatory mechanism of the clock.Recently the rhythmic oscillation of mouse clock genes, mPER1 and mPER2, was shown tobe correlated with the regular alteration of chromatin structure through histoneacetylation/deacetylation. However, little is known about the chromatin modification-mediated transcriptional regulation of Arabidopsis circadian clock genes. Here we proposea possibility that Arabidosis clock-associated genes, LHY, CCA1, and TOC1 might beregulated by rhythmic histone modifications. Our results show that certain type of histonemodifications either has positive or negative correlations with the expression of LHY, CCA1,and TOC1 transcripts. Therefore, the rhythmic transcription of these clock genes mightdepend on regular histone modifications within their chromatin and the fine-tuning of thefeedback loop comprising an oscillator in plants might be accomplished by an orderedmodification of histones.

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Stacey Southall Abstract P110Structural studies of histone methyltransferases

Stacey M. Southall and Jonathon R. Wilson

Chromatin Regulation Team, Section of Structural Biology, Institute of Cancer Research,Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB

Histone lysine methylation is an important epigenetic marker that can profoundly influencedelineation of chromatin regions determining patterns of gene expression and consequentlydefining cell state. This precisely targeted modification is catalysed by a family of histonemethyltransferases (HMTs) containing the evolutionarily conserved SET domain. Our focusis on the methylation of lysine 20 of histone H4. Multiple SET-containing methyltransferasesare able to specifically mono-, di- and/or tri-methylate this lysine residue. Although theprecise downstream effects of these modifications have not been determined, it is clear thateach methylation state has a different physiological role. Structural analysis of SET domainmethyltransferases has given insight into the molecular mechanism of methyltransfer,however to fully understand epigenetic regulation both in the normal cell and in disease weneed to obtain a better understanding of how different HMTs are targeted to the sameresidue and how their activity is regulated at the molecular level.Our strategy is to apply biochemical, biophysical and structural techniques to different H4-K20 HMTs such as nuclear receptor-binding SET domain-containing proteins (NSD family)and the Suv4-20h family. It is now understood that many cancers have an epigeneticcomponent and so modifying enzymes such as HMTs are likely to become drug targets. Ourstudies will therefore help to inform the rational design of such drugs.

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Maike Stam Abstract P111Molecular analysis of chromatin changes involved in b1paramutation, an allele-dependent transfer of epigeneticinformation

Maike Stam, Max Haring, Rechien Bader and Marieke Louwers

SILS, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands

We investigate the molecular mechanism underlying paramutation, a mitotically andmeiotically heritable change in gene expression induced by allele interactions in trans.Paramutation is observed in a wide variety of plants and more recently in fungi andmammals, emphasizing its significance. We study paramutation at the b1 locus in maize,where it affects the pigmentation phenotype. The low expressed B’ epiallele imposes its lowtranscription rate onto the high expressed B-I epiallele in trans. This change correlates withchanges in DNA methylation and chromatin structure.A regulatory element, containing seven tandem repeats located ~100 kb upstream of the b1coding region, is essential for b1 paramutation and functions as an enhancer for the B-Iepiallele. We hypothesize a physical interaction between the repeats and the b1 promoter,and are currently using 3C technology (Dekker et al., 2002) to identify spatial, long range, incis interactions within the 100 kb b1 chromatin domain. The regions spanning the repeatjunctions show differential DNA methylation (only methylated in B’) and nuclease sensitivity(nuclease hypersensitive in B-I), suggesting differential binding of chromatin factors. DNAmethylation analyses of mutants preventing paramutation indicate that the repeat junctionsplay an important role in trans-inactivation, while the regions flanking the junctions play arole in enhancement of b1 expression. ChIP experiments using an anti-H3K4me2 antibodyindicates that both the B’ and B-I coding regions are transcription competent throughoutdevelopment. Upon transcriptional enhancement of b1, the repeat region showsH3K9ac/14ac in high expressing B-I, and H3K9me2 in low expressing B’ tissue, indicating adevelopmentally regulated crosstalk between the transcription activation machinery and theheritable B-I and B’ epigenetic states. Intriguingly, B’ is H3K27 dimethylated at the promoterand coding region throughout development, suggesting a role for H3K27me2 in theheritability of the B’ expression state. We are currently using mutants to dissect the role ofhistone modifications in paramutation.

161

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Sean Taverna Abstract P112Connecting H3 methylation and acetylation : The role ofYng1 in transcription

Sean D. Taverna1, Serge Ilin3, Richard S. Rogers4,5, Jason C. Tanny1,Heather Lavender6, Haitao Li3, Lindsey Baker1, John Boyle4,5, Lauren P.Blair6, Brian T. Chait2, Dinshaw J. Patel3, John D. Aitchison4,5, Alan J.Tackett6 and C. David Allis1

1Laboratory of Chromatin Biology, The Rockefeller University, New York, NY 10021, U.S.A.,2Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University,New York, NY 10021, U.S.A., 3Structural Biology Program, Memorial Sloan-Kettering CancerCenter, New York, NY 10021, U.S.A., 4Institute for Systems Biology, Seattle, WA 98103,U.S.A., 5Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7,Canada, 6Department of Biochemistry and Molecular Biology, University of Arkansas forMedical Sciences, Little Rock, AR 72205, U.S.A.

Post-translational histone modifications participate in modulating the structure and functionof chromatin. Promoters of transcribed genes are enriched with K4 trimethylation andhyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like theYng1 component in the NuA3 HAT complex, were shown to interact with H3K4me3,indicating a biochemical link between K4 methylation and hyperacetylation. Using acombination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD-H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14.Furthermore, genome-wide ChIP-Chip analysis demonstrates co-localization of Yng1 andH3K4me3 in vivo. Disrupting the K4me3-binding capacity of Yng1 altered K14ac andtranscription at certain genes, thereby demonstrating direct in vivo evidence of sequentialtrimethyl-binding, acetyltransferase activity, and gene regulation by NuA3. Our data supporta general mechanism of transcriptional control through which histone acetylation upstreamof gene activation is promoted partially through availability of H3K4me3, “read” by bindingmodules in select subunits.

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Tage Thorstensen Abstract P113The Arabidopsis SUVR proteins define a novel subgroup ofSET domain proteins associated with the nucleolus

Tage Thorstensen, Andreas Fischer1, Silje V. Sandvik, Sylvia S. Johnsen,Paul E. Grini, Gunter Reuter1 and Reidunn B. Aalen

Department of Molecular Biosciences, University of Oslo P.O. Box 1041 Blindern, N-0316 Oslo,Norway, 1Institute of Genetics, Biologicum, Martin Luther University Halle Halle, Germany

The methylation pattern of the different lysine residues on histone tails has been shown tobe part of the so called “histone code” and to be important in the regulation of eukaryoticgene expression and chromatin structure. The proteins responsible for the majority of thismethylation contain the evolutionary conserved SET domain. SET domain proteins relatedto the Drosophila SU(VAR)3-9 protein have been associated with gene repression and hete-rochromatinization. There are 10 SUVH and 5 SUVR genes encoding proteins similar toSU(VAR)3-9 in Arabidopsis, and 4 SUVH proteins have been shown to controlheterochromatic silencing by its HMTase activity and by directing DNA methylation. TheSUVR proteins differ from the SUVH proteins in their domain structure, and we show thatthe closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel WIYLD domain attheir N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescentprotein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus,suggesting involvement in regulation of rRNA expression, in contrast to other SET-domainproteins studied so far which are associated with heterochromatin. The subnuclearlocalization of SUVR proteins is regulated by alternative splicing and we found that theSUVR4 protein is a histone lysine methyltransferase (HKMTase) with preference formonomethylated histone H3K9 in vitro.

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Christopher Topp Abstract P114Unusually-sized centromeric RNAs associate with maizecentromeric chromatin

Christopher N. Topp1 and R. Kelly Dawe1,2

1Department of Plant Biology and 2Department of Genetics, University of Georgia, Athens,GA 30602

Centromeres control the strict inheritance of genetic information, yet are themselvesgenetically ill-defined. The repetitive DNA sequences of most eukaryotic centromeres aredisposed to rapid evolutionary change, differing among even recently diverged species.Currently the best definition for a functional centromere is epigenetic: the presence of acentromere-specific histone variant, CENH3. When highly overexpressed, CENH3 canapparently nucleate de novo kinetochore formation at ectopic locations (Heun et al 2006).However the normal mechanisms that specify CENH3 deposition and the composition ofcentromeric chromatin remain unclear. Here we demonstrate that unusually-sized small,non-coding centromeric RNA species are physically associated with maize centromericchromatin, as assayed by chromatin immunoprecipitation with CENH3. We discuss theimplications of these data for centromere function and evolution.

164



Martin Tribus Abstract P115Molecular mechanisms of histone variant H3.3 assembly bythe motor protein CHD1

Martin Tribus1, Alexander Konev2, Valerie Podhraski1, Dmitry Fyodorov2

and Alexandra Lusser1

1Division of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Insbruck,Austria, 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY10461, U.S.A.

The Drosophila Snf2-related ATPase CHD1 functions as an ATP-dependent chromatinassembly factor. We have previously shown that CHD1 mediates the assembly of extendedperiodic nucleosome arrays in a purified system containing recombinant histone chaperoneNAP-1, purified Drosophila core histones and relaxed circular DNA.We have begun to examine the biological functions of CHD1. To this end, we generatedthree mutant alleles of Chd1 in Drosophila. Two mutations result in a loss of CHD1 protein,while the third allele gives rise to a C-terminally truncated polypeptide. We discovered thatall three alleles are maternal effect embryonic lethal mutations as embryos laid byhomozygous mutant females die before hatching. Intriguingly, we discovered that theabsence of maternal CHD1 blocks the incorporation of the histone variant H3.3 into thepaternal chromatin and thus results in the exclusion of the paternal genome from zygoteformation. Furthermore, CHD1 is required for H3.3 deposition in transcriptionally activechromatin of late syncytial embryos.It was recently shown that the histone chaperone HIRA is required for delivery of H3.3 intothe male pronucleus in Drosophila.Here we present evidence that CHD1 can utilize HIRA as a chaperone to mediate theassembly of H3.3 containing nucleosomes in vitro. We could not detect direct or histonemediated interactions between recombinant CHD1 and HIRA. However, co-immunoprecipitation from Drosophila embryonic extracts revealed a weak association ofCHD1 with HIRA. Thus, our in vivo and in vitro data uncover a CHD1 and HIRA-dependentpathway for the assembly of the histone variant H3.3.CHD1 contains two chromodomains that have recently been shown to be H3-methylK4binding modules. Binding of CHD1 to methylated H3 is thought to be critical for the functionof CHD1 as an elongation factor. To examine the contribution of the chromodomains ofCHD1 to its transcription-independent function in chromatin assembly during earlyembryonic development, we have generated transgenes that carry point mutations atconserved amino acid residues critical for H3-methylK4 binding. We will present data thatshow that the chromodomains are required for some but not all in vivo functions of CHD1.

165

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Christopher Vakoc Abstract P116A profile of histone lysine methylation generated bymammalian gene transcription

Christopher R. Vakoc, Mira M. Sachdeva, Hongxin Wang, and Gerd A. Blobel

Children’s Hospital of Philadelphia,Division of Hematology, Philadelphia, PA 19104, and theUniversity of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.

Complex patterns of histone methylation encode distinct functions within chromatin. Lysinemethylation displays the highest degree of complexity among known covalent histonemodifications, with each site of methylation regulating the association of different effectormolecules. We and others previously reported that tri-methylation of lysine 9 of histone H3occurs at both silent heterochromatin and at the transcribed region of active mammaliangenes, suggesting that the extent of histone lysine methylation involved in mammalian geneactivation is not completely defined. To identify additional sites of histone methylation thatrespond to mammalian gene activity, we describe here a comparative assessment of all sixknown positions of lysine methylation and relate them to gene transcription. For our studies we used the highly expressed housekeeping gene PABPC1 that spans more than 19kb thus permitting high-resolution comparison of histone lysine methylation landscapes. Inaddition, we used genes that can be transcriptionally induced or repressed. We observedhigh trimethylation of H3K4, H3K9, H3K36, andH3K79 in actively transcribed regions,consistent with previous findings. H4K20 mono-methylation, a modification previously linkedwith repression, we identified as a mark of transcription elongation in mammalian cells. Incontrast, H3K27 monomethylation, a modification enriched at peri-centromericheterochromatin, was observed broadly distributed throughout all euchromatic sitesanalyzed, with selective depletion in the vicinity of the transcription start site at active genes.Together, these results underscore that similar to other described methyl-lysinemodifications, H4K20 and H3K27 mono-methylation are versatile and dynamic with respectto gene activity, suggesting the existence of novel site-specific methyltransferases anddemethylases coupled to the transcription cycle.

166



Claudius Vincenz Abstract P117Visualizing polycomb group protein interactions with histonesin vivo

Claudius Vincenz and Tom Kerppola

1150 West Medical Ctr. Dr., 4574 MSRBII, Ann Arbor, MI 48109, U.S.A.

The Polycomb Group proteins consist of about three dozen structurally unrelated proteinsthat were originally identified by their ability to produce a common phenotype in Drosophila.More recent mechanistic studies have identified chromatin as the target for the activity ofthese proteins. Specifically, these proteins both methylate lysine 27 of H3 as well as bind tothis post-translational modification to repress transcription of the underlying DNA. Thisrepression is maintained through cell division producing a molecular memory that enablesthe cell to have a stable phenotype.In Drosophila the Polycomb protein has been shown to be the anchor that recruits a wholecomplex of Polycomb Group proteins to the methylated lysine. In mammalian cells a geneexpansion has produced five homologues of this anchor protein. Here we show that thesestructural homologues, termed CBX proteins in vertebrates, display a nuclear distributionpattern characteristic for each protein. This suggests that methylation of lysine 27 cannotbe the only determinant of their binding to chromatin. Furthermore, we use BimolecularFluorescence Complementation (BiFC) to document that homologous domains havedifferent roles in mediating Histone H3 binding of each CBX protein. For example, deletionof the chromodomain of CBX4 reduces interaction with H3 greatly, confirming in vitrostudies. However, the homologous mutation in CBX6 and CBX7 does not affect H3 binding.Both, CBX6&7 are highly expressed in Embryonic Stem cells. These results are discussedin the context of the unusual distribution of H3K4 and H3K27 methylation in the genome ofES cells. We postulate that this atypical binding mode has evolved to accommodate therapid genome wide changes in transcription that are induced upon differentiation in ES cells.

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Vikki Weake Abstract P118The SAGA histone acetyltransferase complex functions in thedevelopment of neuronal connectivity in the Drosophilacompound eye

Vikki M Weake, Kenneth K. Lee, Sebastian Guelman and Jerry L. Workman

Stowers Institute for Medical Research, Kansas City, Missouri, U.S.A.

The SAGA histone acetyltransferase complex is involved in transcriptional regulation via thecovalent modification of histones. A proteomics approach is being used in our lab to identifyand characterize the components of Drosophila SAGA. This approach has identified twonovel components of the Drosophila SAGA complex: nonstop and CG13379. Nonstop ishomologous to yeast Ubp8, which catalyzes removal of the ubiquitin group from histoneH2B. A critical balance in the level of ubiquitylated H2B is required for correct transcriptionof SAGA regulated genes in yeast, and crosstalk between histone ubiquitylation andmethylation has been observed in previous studies. CG13379 is homologous to yeastSgf11, which together with Ubp8 constitutes a functional module within yeast SAGA. Wehave confirmed that nonstop and Sgf11 are stable components of Drosophila SAGA by co-immunoprecipitation, and a pupal-lethal mutation in sgf11 has been isolated. Furthermore,we show that nonstop can complement the ubp8 deletion strain in yeast and incorporatesinto yeast SAGA. The deubiquitiylation activity of nonstop is currently being characterizedboth in yeast and Drosophila. Independently of our studies, a mutation in nonstop wasisolated during a screen for mutations disrupting neuronal connectivity in the Drosophilavisual system. In order to determine whether the neuronal defect of the nonstop mutant isdue to its role in SAGA, neuronal connectivity of other Drosophila SAGA mutants has beenanalyzed. Mutation of other components of SAGA results in an identical phenotype to thatof nonstop, indicating that SAGA function is required for correct axon targeting in thedeveloping fly eye. The eventual aim of this work is to identify the underlying geneticpathways regulated by SAGA that are required for the establishment of correct neuronalconnectivity in the developing Drosophila eye.

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Stephanie Williams Abstract P119Mechanistic insights into promoter chromatin disassembly

Stephanie Williams, Melissa Adkins and Jessica Tyler

UCHSC, Dept. of Biochemistry, RC-1S, Rm 10403, 12801 E. 17th Ave., Aurora, CO80010, U.S.A.

The disassembly of chromatin from promoter regions is a recently discovered mechanismfor regulation of Eukaryotic gene expression. This process is best understood at thebudding yeast PHO5 gene, where the histone H3/H4 chaperone Anti-silencing function 1(Asf1) is essential for removing 3-4 nucleosomes from the PHO5 promoter upon phosphatedepletion - the signal for activation of the PHO5 gene (Adkins et al., Molecular Cell 2004).We now show that Asf1-mediated promoter chromatin disassembly is also required for theactivation of additional yeast genes. To understand the mechanism whereby promoterchromatin disassembly mediates transcriptional activation, we examined factor occupancy invivo. Somewhat surprisingly, promoter chromatin disassembly is not required to enableaccess of the transcriptional activators to the promoter, but is required to enable access bythe general transcription machinery, including TBP and RNA polymerase II. We will alsopresent our recent analyses of the involvement of chromatin remodelers and histoneacetyltransferases in promoter chromatin disassembly during transcriptional activation.

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Jon Wilson Abstract P120Structural studies of SET domain methyltransferases

Jon Wilson

Institute of Cancer Research, Section of Structural Biology, 237 Fulham Road, Chelsea,London, SW3 6JB, U.K.

The methylation of lysine residues on histone tails is a potent signalling mechanism fordefining patterns of gene expression via variation of chromatin organisation. Only a limitednumber of the lysines in histone tails are subject to modification but the possibility of thediscreet addition of either one, two or three methyl groups allows for a high combinatorialpotential. Correctly regulating the activity of histone methyltransferase enzymes is crucial asthe consequence of mis-regulation can effect the expression of a large number of genesdownstream. It is becoming increasingly apparent that epigenetic processes are involved indisease especially cancer. Whether lysine methylation leads to activation or repression iscontext specific. Disease can be caused by either activation of oncogenes or repression oftumour suppressors.Building upon previous analysis on Set7/9[1] and PR-Set7[2] we are establishing a betterunderstanding of the molecular mechanisms involved in SET domain catalysed methyltransfer. The major area of interest is targeting of a particular lysine residue substrate andthe determination of activity in terms of how many methyl groups a particular enzyme canadd. This is a complex area given that some SET domain methyltransferase enzymes seemto have varying specificity depending on interactions within the multi-protein complexes ofwhich they are components.

Refs:[1] Xiao, B., Jing, C., Wilson, J.R., Walker, P.A., Vasisht, N., Kelly, G., Howell, S.A., Taylor,I.A., Blackburn, G.M. & Gamblin, S.J. (2003) Structure and catalytic mechanism of thehuman histone methyltransferase SET7/9. Nature 421, 652-656.[2] Xiao,B., Jing, C., Kelly, G., Walker, P.A., Muskett, F.W., Frenkiel, T.A., Martin, S.R., Sarma,K., Reinberg, D., Gamblin, S.J. and Wilson, J.R. (2005) Specificity and Mechanism of theHistone Methyltransferase Pr-Set7. Genes & Development 19, 1444-1454.

170



Zhaodong Xu Abstract P121Remote elements critical for cytokine induced gene expression

Zhaodong Xu, Zuyao Ni, Mohamed Abou El Hassan, Tao Yu, MonikaSangwan, Mohamad Ahmad and Rod Bremner

Toronto Western Research Institute, Toronto Western Research Institute, 399 BathurstStreet, Mc6-424,Toronto, Ontario M5T 2S8, Canada

The role played by long-range elements in the regulation of gene transcription is stillunderappreciated. Hunting for these elements is a daunting task and developing a rapid andeasy procedure is essentially important. Towards this end, we utilized histone acetylation toprecisely mark interferon gamma (IFNgamma) specific regulatory elements throughoutmegabase (Mb) regions around 56 known IFN-responsive genes. IFNgamma inducedSTAT1 and IRF1 binding specifically at promoter proximal and remote sites from gene starts.Remarkably the vast majority of IFNgamma-induced STAT1 and IRF1 binding at promotersas well as remote sites was flagged by acetylated histones, verifying the role of acetylatedchromatin in the prediction of promoters and most importantly of long-range elements. Theactivity at novel long-range elements, residing at as far as 70 kb of gene starts, wereconfirmed at the CIITA and SOCS1 loci using ChIP, reporter and looping assays. Thus, long-range elements are a novel aspect of IFNgamma-mediated gene induction and mappingacetylated chromatin is an excellent tool to find them, implying the specificity of the presentmethod in finding remote regulatory elements in large genomic scales.

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Xiaofang Yang Abstract P122Dissecting SWI/SNF ATP-dependent chromatin remodelingcomplex in Saccharomyces cerevisiae

Xiaofang Yang1, Roser Zaurin2, Sharmistha Kundu1, Miguel Beato2 andCraig Peterson1

1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester,MA 01605. 2Centre de Regulacio Genomica, Universitat Pompeu Fabra, Passeig Maritim 37-49, E-08003 Barcelona, Spain

Yeast SWI/SNF is the founding member of an ATP-dependent chromatin remodelingsuperfamily involved in transcriptional regulation for a subset of genes. Most SWI/SNF-likechromatin remodeling enzymes have been purified as multiprotein complexes that containan ATPase catalytic subunit highly homologous to yeast Swi2/Snf2. The fact that ATPasesubunit like human Brg1 alone is active for chromatin remodeling raised the question on therole of other subunits for chromatin remodeling. Interestingly, mutations of core subunits ofhSWI/SNF such as hSwi2/BRG1, hSnf5/INI1, or hSWI3 are found in many types of cancers.In this study, we investigated the role of different subunits for yeast SWI/SNF functionthrough a partial deletion of the SANT domain of Swi3. Genome-wide transcriptional profilesuggested that SWI3deltaSANT was basically a weak null allele of SWI3. SWI3deltaSANTcrippled the recruitment of SWI/SNF to target promoter of CDC6, SIC1 and HO. In theabsence of an intact SANT domain, tethering SWI/SNF via LexA-Swi2 was insufficient fortranscriptional activation of a LexAop-GAL1TATA-LacZ reporter in vivo. Surprisingly,SWI3deltaSANT, SWI3R564E or swi3 caused dissociation of SWI/SNF into at least 4 stablesubcomplexes, indicating that the SANT domain of Swi3 is critical for SWI/SNF assembly.Intriguingly, a triplex of Swi2/Arp7/Arp9, the minimal complex of SWI/SNF, was completelycompetent for ATP hydrolysis, generating superhelical torsion, inducing restriction enzymeaccessibility and catalyzing nucleosome mobility change in vitro. However, the minimalcomplex was defective to catalyze histone H2A/H2B dimer loss from MMTVmononucleosomes. Finally, we found that the minimal SWI/SNF was functional to regulateSRG1 transcription in vivo, based on Northern blot and ChIP. We propose that SWI/SNF isan integration of at least 4 functional modules that are responsible for multi-step chromatinremodeling process. Our data help to understand both structural and functional organizationof SWI/SNF ATP-dependent chromatin remodeling complex.

172



Juan I. Young Abstract P123Post-transcriptional functions of MeCP2

Mauricio A. Saez, Matias Alvarez-Saavedra, Huda Y. Zoghbi and Juan I. Young

Centro de Estudios Cientificos and Universidad Austral de Chile, Valdivia, 905-9100, Chile.Department of Molecular and Human Genetics, Department of Neuroscience and HowardHughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, U.S.A.

Epigenetic modifications of DNA provide an extra level of genetic information. The mainepigenetic modification of mammalian genomes is cytosine methylation, commonlyassociated with gene silencing. The importance of this process for chromatin function hasbeen underscored by the discovery that Rett syndrome, a disabling neurodevelopmentaldisease, is caused by mutations in MeCP2. MeCP2 (methyl-CpG-binding protein 2) is amethylation-dependent transcriptional repressor. We found that MeCP2 is a multifunctional-multilevel regulator of gene expression; interacts with proteins involved in RNA processingand regulates alternative splicing in addition to its role as a transcriptional repressor.

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Veronica Yu Abstract P124Over-expression of Cks proteins causes gene derepressionin Saccharomyces cerevisiae

Roman Holic and Veronica Yu

MRC Clinical Sciences Centre, Imperial College Hammersmith Campus, Du Cane Road,London, W12 0NN, U.K.

Cks (cyclin-dependent kinase interacting) proteins are evolutionarily conserved and arefrequently over-expressed in multiple cancers of high grade and stage.It has previously been demonstrated that the Saccharomyces cerevisiae cell cycleregulatory cyclin-dependent kinase interacting protein, Cks1 and its kinase partner, Cdc28(Cdk1); are involved in the control of transcription at multiple gene loci. This transcriptionalrole is mediated through a requirement of Cdc28/Cks1 for recruiting proteasomes to codingregions. However, it is independent of the protein kinase activity of Cdc28.We over-expressed yeast and mammalian Cks proteins in S. cerevisiae in order to mimicthe protein over-expression pattern in tumour cells. We found that over-expression of Cksproteins caused derepression of a wide-range of genes under repressive conditions.Possible mechanisms and implications for this observation will be discussed.

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Rebekah Zinn Abstract P125hTERT is expressed in cancer despite promoter DNAmethylation by preservation of unmethylated DNA and activechromatin around the transcription start site

Rebekah L. Zinn1,2, Kevin Pruitt1, Sayaka Eguchi1, Stephen B. Baylin1,2 andJames G. Herman1,2

1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland21231 2The Graduate Program in Cellular and Molecular Medicine, The Johns HopkinsUniversity School of Medicine, Baltimore, Maryland 21231

hTERT, which encodes the catalytic subunit of telomerase and is expressed in mostimmortalized and cancer cells, has been reported to have increased DNA methylation in itspromoter region in many cancers. This pattern is inconsistent with observations that DNAmethylation of promoter CpG islands is typically associated with gene silencing. Here weprovide a comprehensive analysis of promoter DNA methylation, chromatin patterns, andexpression of hTERT in cancer and immortalized cells. Methylation specific PCR (MSP) andbisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells showsthat all cancer cell lines retain alleles with little or no methylation around the transcriptionstart site, despite being densely methylated in a region 600 bp upstream of the transcriptionstart site. By real-time RT-PCR, all cancer cell lines express hTERT. Chromatinimmunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9, dimethyl-H3K4)and inactive (trimethyl-H3K9, trimethyl-H3K27) chromatin marks are present across thehTERT promoter. However, using a novel approach combining methylation analysis of ChIPDNA, we show that active chromatin marks are associated with unmethylated DNA, whileinactive marks of chromatin are associated with methylated DNA in the region around thetranscription start site. These results demonstrate that DNA methylation patterns of thehTERT promoter (-150 to +150 around the transcription start) are not inconsistent with theusual dynamics of gene expression in that the absence of methylation in this region and theassociation with active chromatin marks allow for the continued expression of hTERT.

175

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Yoshimitsu Takahashi Abstract P126Degree of SUMO modification as a differential tag for targetingto specific chromosomal domains

Yoshimitsu Takahashi and Alexander Strunnikov

NIH, NICHD, LGRD, Bethesda, MD, U.S.A.

SUMO is a ubiquitin-like modifier that regulates many proteins by direct conjugation. E1, E2and E3 enzymes are needed to transfer SUMO to a specific target. Recently, numerouspotential SUMO substrates were identified by proteomic approaches. However, functionalroles of individual modification are obscure, as only a small fraction of a given protein issumoylated. In order to overcome this technical difficulty, we designed a novel techniqueConstitutive SUMO Modification (CSM), which allows to track only the modified form of theprotein. We validated this method using Top2p as a model substrate. The advantages ofTop2p for application of this technique are: (1) sumoylation sites are in a non-essential C-terminal domain. (2) Top2p expression is abundant. (3) Top2 modification is relatively strongboth in vivo and in vitro. We have evidence that sumoylation of Top2p to different degreeschanges intranuclear targeting of this essential protein.

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Additional poster submissions


Marna S. Costanzo Abstract P127The evolutionary conservation of chromatin modifyingproteins in malaria

Marna S. Costanzo1, Szymon Kaczanowski2, Thanat Chookajorn3, andDaniel L. Hartl1

1Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA02138, USA; 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; 3Department of Biochemistry, Faculty of Science, Mahidol University,Bangkok 10400, Thailand

The malaria parasite is responsible for most of the deaths related to infectious disease inthe world today. This single cell eukaryote has a complex life-cycle consisting of two hostsand many morphologically different forms. The majority of genes expressed during thepathogenic bloodstages correlate to the cell cycle. Transcription of these genes is achievedmonocistronically despite the apparent lack of specific transcription factors and covalenthistone modification provides a possible mechanism of expression control. We investigatethe apparent conservation of histone lysine methyltransferase function in malaria, despitean evolutionary history that is far diverged from most eukaryotes. The implications of thesefindings are discussed in context of the parasite biology and evolutionary implications.

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Abstracts - Poster


Philippe Prochasson Abstract P128

Functional characterization of the HIR corepressor complex

Philippe Prochasson, Laurence Florens and Jerry L. Workman

The Stowers Institute for Medical Research, Kansas City, Missouri 64110, U.S.A.

The histone regulatory (HIR) and histone promoter control (HPC) repressor proteinsregulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle.Previously, we showed that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIRcorepressor complex. The HIR complex promotes histone deposition onto DNA in vitro andconstitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNAand nucleosomes. Furthermore, we demonstrated that the HIR complex binding tonucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF.Thus, the HIR complex is a novel nucleosome assembly complex which functions withSWI/SNF to regulate transcription.We are now pursuing the functional characterization of the HIR complex to better understandits role in gene regulation and its interplay with the SWI/SNF chromatin remodeling complex.We will present data showing that the HIR complex is directly involved in the transcriptionalregulation of the SUC2 gene and that the presence of the HIR proteins at the SUC2promoter renders its transcriptional activation swi2/snf2 dependent. In a second part, we willfocus on the identification of post-translational modifications (PTMs) of the HIR proteinsduring the cell cycle and their role on the histone genes transcriptional regulation.

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Sponsor of 2006 Chromatin Structure & FunctionKeynote Presentation

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Documents

Chromatin Program & Abstract BookStructural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase ... Epigenetic aspects of lineage commitment