P1254413611ANGZe

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Introduction in Epigenetic regulation by
Polycomb and trithorax group proteins
Giacomo CAVALLI. Montpellier, December 2006
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The chromatin Yin / Yang
Euchrochromatin
Heterochromatin

• - Less condensed
• - Gene rich
• Active genes
• Early replicating
• DNA hypomethylated
• - Poor in histone H1
• Histones have specific, activating post
  translational marks

• - Heavily condensed
• - Gene poor
• Silent genes
• Late replicating
• DNA hypermethylated
• - Rich in histone H1
• Histones have repressive post translational marks

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Where on the chromosomes are condensed and open
chromatin located?
Condensed chromatin
Open chromatin
Constitutive Heterochromatin
Typical of the active gene loci along the
chromosomal arms
Telomeres
Centromeres
Condensed chromatin is also found at several
silent genes located in the chromosomal arms
Polycomb group and trithorax group genes are
important regulators of chromatin along the
chromosomal arms
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Polycomb (PcG) and trithorax (trxG) group
proteins epigenetic regulators of genome function

• Originally discovered in Drosophila as regulators
  of Homeotic genes, responsible for specification
  of the body plan, they also regulate many other
  targets involved in cell differentiation and
  proliferation
• PcG proteins silence genes, trxG proteins
  activate them
• Conserved throughout evolution
• In human, they maintain the fates of
  differentiated cells, but they are also required
  for cell proliferation and the maintenance of
  several types of stem cells including ES cells.
  Finally, they regulate X-inactivation in females
  as well as genomic imprinting
• Mutations in PcG and trxG genes induce many
  different cancers

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PcG, trxG, and maintenance of gene expression
Early development
OFF
Maternal, Gap, Pair-rule, Segment polarity
ON
Establishment of patterns
Ubx
ON
OFF
Maintenance phase
trithorax-Group
Polycomb-Group
Transmission of pattern after disappearance of
early factors
ON
OFF
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PcG proteins bind to specific DNA elements, named
PREs
Bound by PcG proteins in vivo (in polytene
chromosomes and by cross-linking experiments)
Binding leads to maintenance of PcG-dependent
repression of reporter genes
Repression is enhanced by the presence of
multiple PRE copies
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Members of the PcG and of the trxG
PcG
protein motifs
Gene
PhoRC Complex
Pleiohomeotic (pho)
Zinc-finger (DNA binding)
Enhancer of zeste (E(z))
SET (H3K27MTase)
Esc/E(z) Complex
extra sex combs (esc)
WD repeat
Polycomb (Pc)
chromo domain (Binding to H3 methyl K9 or K27)
PRC1 complex
polyhomeotic (ph)
Zinc finger, SAM domain
Posterior sex combs (Psc)
RING finger
trxG
protein motifs
Gene
FACT complex
Trithorax-like (Trl)
BTB/POZ (dimerization)
Zinc finger (DNA binding)
TAC1 complex
trithorax (trx)
SET (H3K4HMTase)/ PHD-finger
Ash-1
SET (H3/H4HMTase)/ PHD-finger
bromo domain
Brm complex
brahma (brm)
(DNA dependent ATPase/helicase)
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Action of PcG and trxG complexes on chromatin
trxG
Nucleosome remodeling (BRM complex)
ON
Ac
Maintenance of active states (open chromatin)
Histone acetylation and methylation (TAC1 and
ASH1 complexes)
Me K4 H3
Target gene
PRE
PcG
OFF
Me K27 H3
Deacetylation and methylation (ESC-E(Z) complex)
Maintenance of repressed states (compact
chromatin)
- Chromatin compaction - H2A Ubiquitination (PRC1
complex)
Ub H2A
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Histone H3 K27 methylation and Polycomb
Merge
Pc
H3K27me3
Data from Ringrose et al. (2004) Mol. Cell 16,
641
There is a strong correlation between
trimethylation of K27 (and K9) trimethylation and
Polycomb recruitment at target loci.
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What does Polycomb do to chromatin ? Chromatin
Condensation
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
Data from Francis et al. (2004), Science 306,
1574
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Features of PREs and TREs, and examples of how
they are studied in Drosophila (No PREs
characterized yet in vertebrates)
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1. Example of PcG-dependent spatial specific
silencing of homeotic genes
bxd5.1 UbxlacZ reporter construct
Bxd 5.1 PRE
Ubx prom
LacZ
mini-white
Silencing of a Ubx-lacZ reporter mimicking the wt
behaviour of the Ubx gene, which is silenced in
parasegments 1 to 5
PcG dependent derepression of a Ubx-lacZ reporter
in embryonic territories where it is normally
silenced
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.
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2. Silencing of a transgenic reporter gene
depends on PcG and trxG proteins
Fab-7 Pc /
Fab-7 Pc -/
Fab-7 trx /
Fab-7 trx -/
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4. Recruitment of PcG and trxG proteins to PREs
analysis by ChIP
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PH binding profiles at the embryonic
developmental stage
Negre N, Hennetin J, Sun LV, Lavrov S, Bellis M,
White KP, Cavalli G. Chromosomal Distribution of
PcG Proteins during Drosophila Development. PLoS
Biol. 2006 Apr 204(6)e170
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Example of a high-resolution description of PcG
binding using ChIP and oligonucleotides arrays
with one oligo every 100 bp
H3K27me3
PC
PH
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PcG proteins are required for the maintenance of
ES cell fate, as well as for the fate of
hematopoietic and neuronal stem cells and of
differentiated cell types
Genome-wide Chip on chip in ES cells
Transcription factor family members occupied by
the PRC2 protein SUZ12 in human ES cells
Overlap between PRC1 and PRC2 targets in mouse ES
cells
HOX
Eed (831)
Rnf2 (1219)
IRX
BHLHB
CDX
PRC2
PRC1
MEIS/EVX
66
365
TBX
94
55
DLX
MYO
Suz12 (1271)
Phc (922)
300
164
HES
FOX
ATOH
512
NEUROD
EBF
98
6
GATA
121
31
POU
RUNX
23
PAX
SOX
NKX
LHX
SIX
Lee et al. Cell. 2006 Apr 21125(2)301-13
Boyer et al. Nature. 2006 May 18441(7091)349-53.
Epub 2006 Apr 19
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5. Cross-talk between multiple copies of PREs
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Pairing Sensitive Silencing enhanced silencing
of the mini-white reporter gene by multiple PRE
copies
Chromosome X
Transgenic Fab-7 heterozygous
Transgenic Fab-7 homozygous
w
w
w
---gt strong mini-white silencing
---gt weak silencing of the mini-white reporter
gene
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PcG-mediated silencing is enhanced by the
presence of multiple copies of homologous PREs in
the genome
Fab-X Fab71
Fab-X
sd
sd
Fab-7 transgene
X
X
BX-C
BX-C
Endogenous Fab-7
III
III
Endogenous Fab-7 deletion, named Fab-71
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.
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Homologous Fab-7 copies associate in the nucleus
Dapi
Sd
BX-C
Merge
WT
sd
of interaction
-
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BX-C
Fab-7
-
20
Fab-X
sd
Fab-7
-
15
BX-C
Fab-7
-
10
Fab-X Fab-71
-
5
sd
Fab-7
-
BX-C
0
Fab-X
Fab-X Fab-71
WT
3mm
-


Transgenic Fab-7 at sd (Chr.X)


-
Endogenous Fab-7 at the BX-C (Chr.III)
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Two identical Fab-7 can interact in the nucleus,
leading to an increase of PcG-dependent silencing
Nucleus in a Rabl configuration
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For more info
http//www.igh.cnrs.fr/equip/cavalli http//www.e
pigenome-noe.net In french Inserm Actualités
N. 201, Octobre 2006 (http//www.inserm-actualites
.fr/index.php?id562)
Giacomo.Cavalli_at_igh.cnrs.fr