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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
PRC2
E(z):
Su(z)12:
HMT H3K27me
enhancer of zeste
Zn finger protein
suppressor of zeste; E(z) cofactor
ESC/EED: WD40 protein interaction domains
specificity of HMT; E(z) cofactor
extra sex combs
P55: RbApAB46/48/NURF55 (CAF subunit; sometimes
complexed with HDACs)
-------------------------------------------------------PCL: polycomb like; PhD and Tudor domains
Stimulates 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 lineage
and 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)
PRC1
Pc:
polycomb
has N-terminal chromodomain
binds me H3K27
PH:
polyhomeotic
Zn- finger, Q-repeats
Psc: posterior sex combs (human: Bmi1)
Ring finger protein interaction domain
acts also as suppressor of telomeric position effect
dRING: Ubiquitin ligase H2A K119ub
Simon and Kingston, Nature Reviews Mol Cell Biol, 2009
Two step process?
PRC1 is also recruited independently of H3K27me3
PRC1 occupies large domains, stable repression
Lund and Lohuizen COCB16 239-246 (2004)
PRC1 functions
Inhibits transcription
Blocks SWI/SNF chromatin remdoleing
Compacts chromatin arrays
PRC1-like
Second complex: Ubiquitylation of H2A?
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
Simon and Kingston, 2009
Organism complexity, response to the environment
PRE
PcG Response Element
cis-acting DNA elements that PcG proteins bind to
in 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 binding
Less (but still considerable) overlap with
GAF and DSP1
Binding site mutations: Pho and GAF contribute.
Drosophila
PRC1
PRC2
PRC1
PcG recruitment
Noncoding RNAs can recruit PRC2
PhoRC binds PRE & can recruit PRC2
result: H3K27me
H3K27me can recruit PRC1
PhoRC binds PRE & can recruit PRC1
PREs are devoid of nucleosomes
PhoRC 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 regulatory
regions
Blocks enhancers and binding site PcG!
Deletion causes homeotic phenotypes
Is regulated, allows is tissue/stage specific
enhancer blocker
Developmentally regulated
enhancer blockers
Molecular Cell, Vol. 8, 1145–1151, November, 2001,
HOX genes turned on in certain regions
PcG maintain this pattern (memory)
Also required for proper spatial regulation
later in development
How does the silencing memory work?
PcG required continuously (adult)
maintained through cell cycle
most PcG leaves chromatin during mitosis
some remains = mark?
H3K27 = mark?
PRE = mark?
Also associated with
histone deacetylation
other (H3K9) histone methylation
DNA methylation
memory
PhoRc
Noncoding RNA
Mechanism for PcG repression
Generates large H3K27me domains
Compacts chromatin
Prevents PolII elongation
Form PcG bodies (subnuclear silencing
compartments)
Recruits HDACs, DNMTs
Inhibits 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 flies
suppress ectopic HOX gene expression
Identified trithorax group (TrxG) proteins
Simon and Kingston, 2009
TrxG
Brahma: SWI/SNF ATPase
Moira
Osa
Ash1, 2
Trithorax (TRX)/MLL
Identified as suppressors of PcG mutations
TrxG
TRX: H3K4 KMT
ASH1:
H3K4-(me)3:
H3K4 KMT, also H3K36
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 initiation
Induced by maternal gene products and
segment identity gene products in 3.5 hr embryo
Transcription maintenance
5-7 hr embryo: activators are gone
TrxG maintenance of activated state of homeotic genes
PcG 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 memory
Both can be reversed; needs multiple steps/cues
Generally 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 proteins
MLL plus H3K27 demethylase
chromatin remodeling