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P olyc omb G roup PcG Regulator dentified in Drosophila genetically utations in PcG proteins cause ctopic expression of homeotic genes ANT-C and BX-C (Antennapedia and Bithorax Complex)

Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

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Page 1: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Polycomb Group PcG Regulators

Identified in Drosophila genetically

Mutations in PcG proteins cause ectopic expression of homeotic genes

ANT-C and BX-C(Antennapedia and Bithorax Complex)

Page 2: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Homeotic mutations transcriptional regulation defects

Mutant: T3 haltere into T2 wing

Page 3: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PcG

Three complexes

PRC2

PRC1

PhoRC

Page 4: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 5: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRC2E(z): HMT H3K27me

enhancer of zeste

Su(z)12: Zn finger proteinsuppressor of zeste; E(z) cofactor

ESC/EED: WD40 protein interaction domainsspecificity of HMT; E(z) cofactor

extra sex combs

P55: RbApAB46/48/NURF55 (CAF subunit; sometimes complexed with HDACs)--------------------------------------------------------PCL: polycomb like; PhD and Tudor domainsStimulates enzyme activity, role in recruitment?

Page 6: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

Page 7: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRC2 unique roles

Required for X-inactivation, imprinting

Stem cell maintenance

EZH2 (human E(z)) important marker for tumor metastasis)

Page 8: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 9: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRC2 is required for Xi in the extra-embryonic lineageand in the embryo

puts on H3K27me mark

occurs right after Xist coating

Xist is necessary and sufficient for PRC2 recruitment

Page 10: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRC2 recruitment by long nc RNAs

XIST (X-inactivation)

Kcnq1ot1 (Imprinting)

Hotair (Hox gene expression)

Page 11: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 12: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Pc: polycombhas N-terminal chromodomainbinds me H3K27

PH: polyhomeoticZn- finger, Q-repeats

Psc: posterior sex combs (human: Bmi1)Ring finger protein interaction domainacts also as suppressor of telomeric position effect

dRING: Ubiquitin ligase H2A K119ub

PRC1

Page 13: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

Page 14: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Lund and Lohuizen COCB16 239-246 (2004)

Two step process?

PRC1 is also recruited independently of H3K27me3PRC1 occupies large domains, stable repression

Page 15: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRC1 functions

Inhibits transcription

Blocks SWI/SNF chromatin remdoleing

Compacts chromatin arrays

Page 16: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Second complex: Ubiquitylation of H2A?

PRC1-like

Simon and Kingston, 2009

Page 17: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 18: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PhoRC

Pho: DNA binding protein

SFMBT: can bind to certain methylated lysines on H3

Page 19: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

YY1

Page 20: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Mammals (and plants)Many PRC2 and PRC1 complexes

Organism complexity, response to the environment

Simon and Kingston, 2009

Page 21: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PRE

PcG Response Element

cis-acting DNA elements that PcG proteins bind toin Drosophila

Many PREs in homeotic gene regulatory regions

PREs are comprised of many factor biding sites

Page 22: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PREs have many binding sites for certain TFs

Page 23: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PREs

Genomewide studies:PRC1 binding largely overlaps with Pho bindingLess (but still considerable) overlap with GAF and DSP1

Binding site mutations: Pho and GAF contribute.

Page 24: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Drosophila

PRC1

PRC1

PRC2

Page 25: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PcG recruitment

Noncoding RNAs can recruit PRC2PhoRC binds PRE & can recruit PRC2

result: H3K27me

H3K27me can recruit PRC1PhoRC binds PRE & can recruit PRC1

PREs are devoid of nucleosomesPhoRC can wrap DNA around itself

in presence of PRC1

Page 26: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Simon and Kingston, 2009

Page 27: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 28: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 29: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

HOX gene regulation

Normal expression

Page 30: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PREs have many binding sites for certain TFs

Page 31: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Fab-7

Enhancer blocker between iab-6 and iab-7 regulatoryregions

Blocks enhancers and binding site PcG!

Deletion causes homeotic phenotypes

Is regulated, allows is tissue/stage specific enhancer blocker

Page 32: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Molecular Cell, Vol. 8, 1145–1151, November, 2001,

Developmentally regulatedenhancer blockers

Page 33: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

HOX genes turned on in certain regions

PcG maintain this pattern (memory)

Also required for proper spatial regulationlater in development

Page 34: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

How does the silencing memory work?

PcG required continuously (adult)maintained through cell cyclemost PcG leaves chromatin during mitosissome remains = mark?

H3K27 = mark? PRE = mark?

Also associated with histone deacetylation

other (H3K9) histone methylationDNA methylation

memory

Page 35: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PhoRcNoncoding RNA

Page 36: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Mechanism for PcG repression

Generates large H3K27me domains Compacts chromatin Prevents PolII elongationForm PcG bodies (subnuclear silencing compartments)Recruits HDACs, DNMTsInhibits SWI/SNF activity

Page 37: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Science 308 (2005)

Page 38: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

mammals

Page 39: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Drosophila

Page 40: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Block of transcription elongation?

Simon and Kingston, 2009

Page 41: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

http://www.igh.cnrs.fr/equip/cavalli/Figure0.jpeg

Page 42: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Supressor screen in polycomb mutants

Look for wild-type looking fliessuppress ectopic HOX gene expression

Identified trithorax group (TrxG) proteins

Page 43: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Simon and Kingston, 2009

Page 44: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

TrxG

Brahma: SWI/SNF ATPaseMoiraOsa

Ash1, 2

Trithorax (TRX)/MLL

Identified as suppressors of PcG mutations

Page 45: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

TrxG

TRX: H3K4 KMT

ASH1: H3K4 KMT, also H3K36

H3K4-(me)3: TrxG binding inhibits PcG binding inhibits HP1 binding

Page 46: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PREs have many binding sites for certain TFs

Page 47: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Often PcG and TrxG bind the same element

PRE

Continuous inactivation required for PcG silencing

Transcription through PRE causes loss of silencing

Page 48: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Homeotic Gene Expression

Transcription initiationInduced by maternal gene products and segment identity gene products in 3.5 hr embryo

Transcription maintenance5-7 hr embryo: activators are goneTrxG maintenance of activated state of homeotic genesPcG maintenance of repressed state of homeotic genes

Page 49: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Mammals

Some PREs recently identified

TrxG and PcG opposing roles conserved

Also in plants

Page 50: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PcG (and TrxG)

Not just transcriptional memory

more dynamic

gets reset

Cell fate not as fixed as was previously assumed

Page 51: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Noncoding RNA

Page 52: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 53: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Not this simple

Page 54: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Opposing roles of TrxG and PcG

Reversibility of marks UTX JMJD3 part of TrxG complex LSD1 and JARID associate with PcG complex

Page 55: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Bivalent domains

Page 56: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 57: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Embryonic stem cells

PcG proteins occupies promoters of

differentiation genes

prevents differentiation

Page 58: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C
Page 59: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Associated with poised polymerase

Page 60: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Some concerns

Contradictory data

Marks not shown to be present at the same locus: population effect?

In some cases no evidence for poised PolII.

Page 61: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

Going further

Both TrXG and PcG stable epigenetic memoryBoth can be reversed; needs multiple steps/cuesGenerally TrxG and PcG have a dynamic role

Lund and Lohuizen COCB16 239-246 (2004)

Page 62: Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C

PcG silencing reversal (needs several steps)

•Resetting by transcription

•Signaling repression of PcG(WNT/TGFB/HH)

•Posttranslational modifications PcG(BMI1-P dissociates)

•Recruitment of TrxG proteinsMLL plus H3K27 demethylasechromatin remodeling