1. The chromatin Yin / Yang
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The chromatin Yin / Yang
Heterochromatin
Euchrochromatin
- Heavily condensed
- Gene poor
Silent genes
Late replicating
DNA hypermethylated
- Rich in histone H1
Histones have repressive post translational marks
- Less condensed
- Gene rich
Active genes
Early replicating
DNA hypomethylated
- Poor in histone H1
Histones have specific, activating post translational marks

2. Where on the chromosomes are condensed and open chromatin located?
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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

3. Chromatin
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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

4. PcG, trxG, and maintenance of gene expression
Chromatin
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Early development
OFF ON
Maternal, Gap, Pair-rule, Segment polarity
Establishment of patterns
Ubx
OFF ON
Polycomb-Group
Maintenance phase
trithorax-Group
Transmission of pattern after disappearance of early factors
OFF ON
Intro

5. Members of the PcG and of the trxG
Chromatin
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Members of the PcG and of the trxG
PcG
Gene
protein motifs
PhoRC
Complex
Pleiohomeotic (pho)
Zinc-finger (DNA binding)
Esc/E(z)
Complex
Enhancer of zeste (E(z))
SET (H3K27MTase)
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
Gene
protein motifs
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
Brm complex
bromo domain
brahma (brm)
(DNA dependent ATPase/helicase)

6. PcG proteins bind to specific DNA elements, named PREs
Chromatin
PcG proteins bind to specific DNA elements, named PREs
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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

7. Action of PcG and trxG complexes on chromatin
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Action of PcG and trxG complexes on chromatin
Nucleosome remodeling
(BRM complex)
trxG ON Ac
Maintenance of active states
(open chromatin)
Histone acetylation and methylation
(TAC1 and ASH1 complexes)
Me K4 H3
Target gene
PRE
OFF
PcG
Me K27 H3
Deacetylation and methylation
(ESC-E(Z) complex)
Maintenance of repressed states
(compact chromatin)
- Chromatin compaction
- H2A Ubiquitination
(PRC1 complex)
Ub H2A

8. What does Polycomb do to chromatin ? Chromatin Condensation
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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

9. Chromatin
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2. Silencing of a transgenic reporter gene depends on PcG and trxG proteins
Fab-7
UAS-lacZ
white
Fab-7
Pc -/+
Fab-7
Pc +/+
Fab-7
trx -/+
Fab-7
trx +/+

10. PcG and trxG proteins associate to multiple genomic loci
Chromatin
PcG and trxG proteins associate to multiple genomic loci PH
DAPI
Polytene chromosome staining shows around 100 bands for each PcG protein
Merge
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11. Chromatin
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Recruitment of PcG proteins at PREs: chromatin analysis by chromatin immunoprecipitation (ChIP) and DNA microarrays (chips)
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 to hybridize DNA chips containing the genome. Extract the distribution profile of the proteins of interest
ChIP on chip

12. Example of the chromosomal distribution of PcG proteins at BX-C locus
Ubx
AbdB
abdA *
bx bxd iab iab4 mcp Fab Fab8
BX-C PH
Suz12
Pcl PC
H3K27me3
E(z)
Example of the chromosomal distribution
of PcG proteins at BX-C locus

13. PcG proteins form nuclear compartments called PcG bodies
Z axis maximum intensity projection
PC / Heterochromatin
We count about PcG bodies in diploid nuclei, while ChIP on chip finds over 200 Polycomb domains
We postulate that multiple Polycomb domains are clustered in the nucleus !!

14. Genome-wide Chip on chip in ES cells
Chromatin
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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
Eed
(831)
Rnf2
(1219)
HOX
IRX
BHLHB
PRC2
PRC1
CDX 66
365
TBX
MEIS/EVX 94 55
DLX
Suz12
(1271)
Phc
(922)
MYO
300
164
HES
FOX
512
ATOH
NEUROD 98 6
EBF
GATA
121 31
POU
RUNX 23
SOX
PAX
NKX
SIX
LHX
Boyer et al. Nature May 18;441(7091): Epub 2006 Apr 19
Lee et al. Cell Apr 21;125(2):301-13

15. Regulation of transcriptional pathways is
a major « raison d’etre » of PcG proteins 48 6 18 26
240 29
Mouse PcG
targets
Human PcG
Fly PcG
Transcription factor
activity (87.4%)
Potassium channel
activity (2.8%)
Morphogen
(2.8%)
Receptor activity
Electron transporter
activity (1.4%)
Unknown
N=72
common targets to fly and vertebrates
Total No. of fly target genes: 260
98 target genes in fly, that have
clear human and mouse homologs
Unknown
(7.7%)
mRNA binding
(11.45%)
Actin binding,
cytoskeleton
(15.4%)
Kinase activity
(3.85%)
Oxidoreductase
activity (3.85%)
Dehydrogenase
Receptor activity
Nucleic acid
binding (3.85%)
coA ligase activity
Alkaline phosphatase
Transcription factor
activity (38.5%)
N=26
unique targets for fly

16. PcG and trxG proteins regulate cell memory and dynamic patterns of gene expression
stem cell maintenance
and plasticity
proliferation
differentiation
PcG and trxG
proteins
X-chromosome inactivation
cell fate
determination
Vertebrates
Fly spermatogenesis
Genomic imprinting
Cancer

17. The ph0 clones show ectopic autonomous mitosis in eye discs
Anti PHOSPHO-H3 wt
ph0 clones
Image ph505ph36 du dans projet2007

18. Massive overgrowth of adult ph0 eyes
Control
Mutant escapers
Dossier annemarie60307 dans projet 2°semestre 2006
90% lethal
10% of escapers
Polyhomeotic is a tumor suppressor gene !

19. Chromatin
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Allez, on a presque fini… une dernière question: alors, elle a quelle forme cette chromatine en fait ?

20. Chromatin
Chromatine active
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21. Chromatin
Chromatine réprimée
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22. Website to download this course:
Chromatin
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For more info…
Inserm Actualités N. 201, Octobre 2006 (
Polycomb and cancer:
Chromatin movie:
Website to download this course: