P oly c omb G roup PcG Regulators

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)

Homeotic mutations transcriptional regulation defects

Mutant: T3 haltere into T2 wing

PcG

Three complexes

PRC2

PRC1

PhoRC

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?

Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

PRC2 unique roles

Required for X-inactivation, imprinting

Stem cell maintenance

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

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

PRC2 recruitment by long nc RNAs

XIST (X-inactivation)

Kcnq1ot1 (Imprinting)

Hotair (Hox gene expression)

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

Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

Lund and Lohuizen COCB16 239-246 (2004)

Two step process?

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

PRC1 functions

Inhibits transcription

Blocks SWI/SNF chromatin remdoleing

Compacts chromatin arrays

Second complex: Ubiquitylation of H2A?

PRC1-like

Simon and Kingston, 2009

PhoRC

Pho: DNA binding protein

SFMBT: can bind to certain methylated lysines on H3

YY1

Mammals (and plants)Many PRC2 and PRC1 complexes

Organism complexity, response to the environment

Simon and Kingston, 2009

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

PREs have many binding sites for certain TFs

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.

Drosophila

PRC1

PRC1

PRC2

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

Simon and Kingston, 2009

HOX gene regulation

Normal expression

PREs have many binding sites for certain TFs

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

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

Developmentally regulatedenhancer blockers

HOX genes turned on in certain regions

PcG maintain this pattern (memory)

Also required for proper spatial regulationlater in development

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

PhoRcNoncoding RNA

Mechanism for PcG repression

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

Science 308 (2005)

mammals

Drosophila

Block of transcription elongation?

Simon and Kingston, 2009

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

Supressor screen in polycomb mutants

Look for wild-type looking fliessuppress ectopic HOX gene expression

Identified trithorax group (TrxG) proteins

Simon and Kingston, 2009

TrxG

Brahma: SWI/SNF ATPaseMoiraOsa

Ash1, 2

Trithorax (TRX)/MLL

Identified as suppressors of PcG mutations

TrxG

TRX: H3K4 KMT

ASH1: H3K4 KMT, also H3K36

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

PREs have many binding sites for certain TFs

Often PcG and TrxG bind the same element

PRE

Continuous inactivation required for PcG silencing

Transcription through PRE causes loss of silencing

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

Mammals

Some PREs recently identified

TrxG and PcG opposing roles conserved

Also in plants

PcG (and TrxG)

Not just transcriptional memory

more dynamic

gets reset

Cell fate not as fixed as was previously assumed

Noncoding RNA

Not this simple

Opposing roles of TrxG and PcG

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

Bivalent domains

Embryonic stem cells

PcG proteins occupies promoters of

differentiation genes

prevents differentiation

Associated with poised polymerase

Some concerns

Contradictory data

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

In some cases no evidence for poised PolII.

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)

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