Polycomb, trithorax, and maintenance of gene expression

A key feature of development in metameric animals is the definition of body segments where groups of cells with a specified fate will give rise to their relative body structures. Cell fates are specified by particular combinations of homeotic gene products. During early embryogenesis, maternal and segmentation genes regulate homeotic genes by binding to specific regulatory sequences located in the promoter regions.  Later in development, the expression pattern of homeotic genes as well as other important developmental genes are maintained by a cell memory system dependent on two groups of genes. The members of these two groups are able to recognize the active and inactive state of expression and fix it to the cell progeny through many cell divisions. These components have been classified in two genetic groups. The trithorax-group (trxG) maintain the active state of expression, while the Polycomb-group (PcG) counteracts this activation with a stable repressive function.

There is strong evidence that the memory function encoded by these two groups of genes is achieved through regulation of higher order chromatin structures. PcG gene products form large multimeric protein complexes in Drosophila, mouse and human. PcG mediated gene silencing can be directed by DNA elements in cis, defined as PcG response elements (PRE). On the other hand, several trxG members act at elements defined as TRE (that overlap with PRE) via chromatin remodeling and induction of histone modifications that increase chromatin accessibility to transcription factors.

In this set of slides, some of the features of PcG and of trxG factors are summarized and examples of molecular and cell biological approaches to dissect their mechanisms of action are presented.

Polycomb, trithorax, and maintenance of gene expressionEarly development Establishment of patterns

Maternal, Gap, Pair-rule, Segment polarityONOFFUbx

Polycomb-Group trithorax-GroupMaintenance phaseTransmission of pattern after disappearance of

early factors

ON OFF

ON OFF

haltere

Ubx OFF Ubx ON

wing Update: December 2004

PcG and trxG proteins associate to multiple genomic loci

PH

DAPI

Polytene chromosome staining shows around 100 bands for each PcG protein

Merge

Binding leads to maintenance of PcG-dependent repression of reporter genes

Bound by PcG proteins in vivo (in polytene chromosomes and by cross-linking experiments)

Repression is enhanced by homologous pairing of the transgenes

Bound by trxG proteins in vivo

Binding leads to maintenance of trxG-dependent activation of reporter genes

PRE and TRE often overlap in the same genomic region

PcG and trxG proteins bind to specific DNA elements, named PRE and TRE

PRE

TRE

protein motif homologs

Members of the PcG and of the trxG

Gene

trithorax (trx) SET (H3 HMTase)/ PHD-finger MLL/ALL-1/HRX (human)

brahma (brm) bromo domain SWI2/SNF2 (yeast)

(DNA dependent ATPase/helicase) brg1 (mouse/human); Hbrm (human)

Trithorax-like (Trl)zinc finger (DNA binding)BTB/POZ (dimerization)

trxG

protein motif homologsGenePcGPolycomb (Pc) chromo domain

(Binding to H3 methyl K9 or K27)

M33 (mouse); hPC (human)

polyhomeotic (ph) one zinc finger Mph1/Rae-28 (mouse); hph1; hph2 (human)

Posterior sex combs (Psc) RING finger bmi-1 (mouse/human); mel-18 (mouse)

Pleiohomeotic (pho) Zinc-finger (DNA binding) hYY-1 (human); mYY-1 (mouse)

Enhancer of zeste (E(z)) SET (H3MTase) Ezh1; Ezh2 (human); clf (Arabidopsis)

extra sex combs (esc) WD repeat Eed (mouse); hEED (human)

PRC1 complex

Esc/E(z)Complex

Ash-1 SET (H3/H4HMTase)/ PHD-finger ASH-1 (human); NSD1 (mouse)

TAC1 complex

Brm complex

FACT complex

OFF

ONtrxG

Maintenance of active states

(open chromatin)

PRETarget gene

Histone acetylation and

methylation(TAC1 and ASH1

complexes)

Deacetylation and methylation

(ESC-E(Z) complex)

Maintenance of repressed states

(compact chromatin)

PcG

- Chromatin compaction

- H2A Ubiquitination(PRC1 complex)

Nucleosome remodeling(BRM complex)

Ac

Me K27 H3

Action of PcG and trxG complexes on chromatin

Me K4 H3

Ub H2A

Histone H3 methylation and PolycombPc

Pc H3 K27 triMe

H3 K9 triMe Merge

Merge

There is a strong but not absolute correlation between trimethylation of K27 (and K9) trimethylation and Polycomb recruitment at target loci. i.e. there is more to Pc

recruitment

Data from: Ringrose et al. (2004) Mol. Cell 16, 641

What do « Polycomb » proteins do to chromatin ?1. Condensation

Data from: Francis et al. (2004), Science 306, 1574

Recombinant PC-containing complexes can condense an array of 12 nucleosomes in vitro

Condensation requires PSC (not PH) protein, and involves histones but does not necessitate histone tails

What do « Polycomb » proteins do to chromatin ?2. H2A Ubiquitination

Purified human PRC1-type complexes can Ubiquitinate H2A in vitro, and the drosophila counterpart of the same complex induces a dRing-dependent H2A Ub at the Ubx PcG target gene

Data from: Wang et al. (2004) Nature 431, 873

Features of PREs and TREs, and examples of how they are

studied in drosophila

The Bithorax-complex is a target locus for PcG and trxG proteins

Fab-7&8iab-4

iab2/3bxdbx

Mcp

Regulatory regions in the Bithorax Complex are shown in red

Target elements for PcG and/or trxG proteins are shown in green

Ubx UbxHbZYG HbZYG

Extended germ band stage (6h)

Ubx Ubx

Gastrulation stage (3h)

Wild type embryo Mutant PcG embryo

---> Anterior derepression, antero-posterior transformation

A P A P

1. Spatial specific maintenance of silencing of homeotic genes

No effect on initiation of silencing in anterior parasegments

Silencing initiates correctly, thanks to early repressors like Hunchback (Hb), but degenerates in the absence of PcG proteins

when these repressors disappear

1. Example of PcG-dependent spatial specific silencing of homeotic genes

PcG dependent derepression of a Ubx-lacZ reporter in embryonic territories where it is normally silenced

Silencing of a Ubx-lacZ reporter mimicking the wt behaviour of the Ubx gene, which is silenced in parasegments 1 to 5

bxd5.1 UbxlacZ reporter construct

Bxd 5.1 PRE Ubx promLacZ mini-white

Data from: Hodgson, J. W., Argiropoulos, B., and Brock, H. W. (2001). Site-specific recognition of a 70-base-pair element containing d(GA)(n) repeats mediates bithoraxoid polycomb group response element-dependent silencing. Mol Cell Biol 21, 4528-4543.

During embryonic development, chromosome homologs pair in diptera. Pairing brings homolog sequences in close physical proximity. This

pairing correlates with the strength of PcG and trxG mediated regulation

Weak PcG mediated repression

Strong PcG mediated repression

PRE Heterozygous PRE Homozygous

2. Pairing Sensitive Silencing

Ubx abd-A Abd-B

3,6 Kb

Locus BX-C

Chr. III

Boundary PRE

The Fab-7 element is a 3.6 Kb region that regulates expression of the homeotic gene Abdominal-B (Abd-B), located in the locus named Bithorax Complex (BX-C) in chromosome III of Drosophila. This element is partitioned in a PRE and a so-called “chromatin boundary”, i.e. an element that might segregate independent chromosomal domains from each other.

Fab-7

The Fab-7 element of the BX-C

w w

w

Chromosome X

2. An example of Pairing Sensitive Silencing: silencing of the mini-white reporter gene by the Fab-7 element in the Fab-X transgenic line

---> strong mini-white silencing---> weak silencing of the mini-white reporter gene

Transgenic Fab-7heterozygous

Transgenic Fab-7homozygous

PP mini-whiteFab-7

Data from: Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G. (2003). Inheritance of Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev 17, 2406-2420.

2. Silencing of mini-white depends on PcG and trxG proteins

Fab-7Pc +/+

Fab-7Pc -/+

Fab-7trx +/+

Fab-7trx -/+

Fab-7 UAS-lacZ white

3. Recruitment of PcG and trxG proteins to PREs: analysis in Fab-7 by a combination of immunostaining and FISH in polytene chromosomes (immuno-FISH)

24A

25E5

transgene

DAPI Immunostainingof PH protein

FISH Immuno-FISH

Fab-7 UAS-lacZ white

Transgene :

4. Recruitment of PcG proteins at PREs: chromatin analysis by Formaldehyde cross-linking and chromatin immunoprecipitation (ChIP)

Sonicate and purify chromatin (average size = 1 kb)

Add antibody and purify antibody-chromatin complexes on Protein A Sepharose, purify DNA and amplify by Linker-mediated PCR

Cross-link cells or embryos with formaldehyde to induce protein-DNA crosslinks

Use amplified DNA as probe on a Southern of a genomic walk, quantify by PhosphorImager

Genomic walk

1230 bp

778 bp

422 bp

356 bp

GAFXhEH H XbXbPF

200

120

40

Boundary PRE

200

120

40

1 Kb

p d

50

30

10

XhEH H XbXbPF

50

30

10

1 Kb

Boundary PRE

PC

p d

GAFPC

Mock PC Ip Mock GAGA Ip

Example: analysis of PC and GAGA factor binding to Fab-7 by ChIP

Quantification of the signals

Data from: Cavalli, G., and Paro, R. (1998). The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93, 505-518.

Fab-7 UAS-lacZ white

Hsp 70 GAL4

Driver construct

Reporter construct

The GAL4 system for the study of PRE/TRE function: mimicking the developmental pathway leading to maintenance of homeotic gene expression

Check Eye colorLight Eye color white repressed

Embryo

Larva

Pupa1st 2nd 3rd instar

HsGAL4 pulse during early development

Experimental approach

?

5. Maintenance of active as well as repressed states: PREs and TREs form elements named as Cellular Memory Modules

GAL4

Fab-7: a cellular memory module (CMM) that maintains active as well as silenced chromatin throughout development

+HS

-HS

lacZ whiteFab-7

G

3,6 kb

Gal4hsp70 GAL4

HS

UAS

Beta-gal stains to study lacZ expression

Data from: Cavalli, G., and Paro, R. (1998). The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93, 505-518.

Chromatin states can be inherited through meiosis by the following

generations

Meiotic inheritance of derepressed states

Repressed Derepressed G0

GAL4 pulse in embryos Re-cross red eyed flies

Derepressed G1, G2, G3...

Big open questions

What are the precise developmental cues that recruit PcG or trxG proteins to PREs of homeotic genes? Do they also apply for other PREs/TREs?

What are the molecular mechanisms for recruitment of PcG and trxG proteins to PREs and TREs? What are the effects of these recruitments on chromatin?

Once recruited, how can these proteins maintain chromatin states through DNA replication and through mitosis (and meiosis)?

What is the basis of the Pairing Sensitive Effects and of long distance interactions? How does this phenomenon contribute to inheritance of chromatin states?

What is the genome-wide profile of PcG and trxG binding? What is the identity of the corresponding target genes? Are they all regulated like homeotic genes, or are there different categories of regulatory mechanisms?