EuchrochromatinHeterochromatin

The chromatin Yin / Yang

- 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

Where on the chromosomes are condensed and open chromatin located?

Condensed chromatin Open chromatin

Constitutive Heterochromatin

Telomeres

Centromeres

Condensed chromatin is also found at several

silent genes located in the chromosomal

arms

Typical of the active gene loci along the chromosomal arms

Polycomb group and trithorax group genes are important regulators of chromatin along the chromosomal arms

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

PcG, trxG, and maintenance of gene expressionEarly development Establishment of patterns

Maternal, Gap, Pair-rule, Segment polarityONOFF

Ubx

Polycomb-Group trithorax-GroupMaintenance phaseTransmission of pattern after disappearance of

early factors

ON OFF

ON OFF

protein motifs

Members of the PcG and of the trxG

GenetrxG

protein motifsGenePcG

Polycomb (Pc) chromo domain(Binding to H3 methyl K9 or K27)

polyhomeotic (ph) Zinc finger, SAM domain

Posterior sex combs (Psc) RING finger

PRC1 complex

Enhancer of zeste (E(z)) SET (H3K27MTase)

extra sex combs (esc) WD repeat

Esc/E(z)Complex

trithorax (trx) SET (H3K4HMTase)/ PHD-finger

Ash-1 SET (H3/H4HMTase)/ PHD-finger

TAC1 complex

brahma (brm) bromo domain

(DNA dependent ATPase/helicase)Brm complex

Trithorax-like (Trl)Zinc finger (DNA binding)BTB/POZ (dimerization)FACT

complex

Pleiohomeotic (pho) Zinc-finger (DNA binding)PhoRCComplex

Binding leads to maintenance of PcG-dependent repression of reporter genes

Bound by PcG proteins in vivo (in polytene chromosomes and by cross-linking experiments)

Repression is enhanced by the presence of multiple PRE copies

PcG proteins bind to specific DNA elements, named PREs

OFF

ONtrxG

Maintenance of active states

(open chromatin)

PRETarget gene

Histone acetylation and methylation

(TAC1 and ASH1 complexes)

Deacetylation and methylation

(ESC-E(Z) complex)

Maintenance of repressed states

(compact chromatin)

PcG

- Chromatin compaction- H2A Ubiquitination

(PRC1 complex)

Nucleosome remodeling(BRM complex)

Ac

Me K27 H3

Action of PcG and trxG complexes on chromatin

Me K4 H3

Ub H2A

What does Polycomb do to chromatin ?Chromatin Condensation

Data from: Francis et al. (2004), Science 306, 1574

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

2. Silencing of a transgenic reporter gene depends on PcG and trxG proteins

Fab-7Pc +/+

Fab-7Pc -/+

Fab-7trx +/+

Fab-7trx -/+

Fab-7 UAS-lacZ white

PcG and trxG proteins associate to multiple genomic loci

PH

DAPI

Polytene chromosome staining shows around 100 bands for each PcG protein

Merge

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

Ubx AbdBabdA

** ****

bx bxd iab2 iab4 mcp Fab7 Fab8

*BX-C

PH

Suz12

Pcl

PC

H3K27me3

E(z)

Example of the chromosomal distributionof PcG proteins at BX-C locus

Z axis maximum intensity projectionPC / Heterochromatin

PcG proteins form nuclear compartments called PcG bodies

We count about 30-50 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 !!

GATA

ATOH

LHX

POU

IRX

DLX

SIX

NEUROD

BHLHB

PAX

FOX

HOX

SOX

TBX

NKX

HES

EBF

RUNX

MYO

CDX

MEIS/EVX

Transcription factor family membersoccupied by the PRC2 protein SUZ12 in human

ES cells

Overlap between PRC1 and PRC2 targets in mouse ES cells

Eed(831)

Suz12(1271)

Rnf2(1219)

Phc(922)

PRC2 PRC166

94

300

98 6

164

55

365

121 31

512

23

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

Lee et al. Cell. 2006 Apr 21;125(2):301-13Boyer et al. Nature. 2006 May 18;441(7091):349-53. Epub 2006 Apr 19

48

6

18

26

240

29

18 Mouse PcGMouse PcGtargets targets

Human PcGHuman PcGtargetstargets

Fly PcGFly PcGtargetstargets

Regulation of transcriptional pathways isa major « raison d’etre » of PcG proteins

Total No. of fly target genes: 26098 target genes in fly, that have clear human and mouse homologs

Transcription factoractivity (87.4%)

Potassium channel activity (2.8%)

Morphogen(2.8%)

Receptor activity (2.8%)

Electron transporteractivity (1.4%)

Unknown(2.8%)

N=72

common targets to fly and vertebrates

Unknown(7.7%)

mRNA binding(11.45%)

Actin binding,cytoskeleton

(15.4%)

Kinase activity(3.85%)

Oxidoreductase activity (3.85%)

Dehydrogenaseactivity (3.85%)

Receptor activity(3.85%)

Nucleic acidbinding (3.85%)

coA ligase activity(3.85%)

Alkaline phosphatase activity (3.85%)

Transcription factoractivity (38.5%)

N=26

unique targets for fly

PcG and trxG proteins

stem cell maintenanceand plasticity

Genomic imprintingGenomic imprintingGenomic imprintingGenomic imprinting

Cancer

proliferation

X-chromosome X-chromosome inactivationinactivationX-chromosome X-chromosome inactivationinactivation

differentiation

cell fate determination

PcG and trxG proteins regulate cell memory and dynamic patterns of gene expression

Fly spermatogenesisFly spermatogenesisFly spermatogenesisFly spermatogenesis

VertebratesVertebrates

The ph0 clones show ectopic autonomous mitosis in eye discsAnti PHOSPHO-H3

wt

ph0 clones

90% lethal10% of escapers

Control

Massive overgrowth of adult ph0 eyes

Mutant escapers

Polyhomeotic is a tumor suppressor gene !

Allez, on a presque fini…une dernière question: alors,

elle a quelle forme cette chromatine en fait ?

Chromatine active

Chromatine réprimée

Giacomo.Cavalli@igh.cnrs.fr

For more info…

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

http://www.epigenome-noe.net

Inserm Actualités N. 201, Octobre 2006 (http://194.254.71.5/basisdiaggen/IA/201/Mag201-dossier.pdf)

Polycomb and cancer: http://www2.cnrs.fr/presse/communique/1672.htm

Chromatin movie: http://www.dailymotion.pl/tag/nisic

Website to download this course:http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html