CavalliCavalli LabLab

Introduction in:Epigenetic regulation by Polycomb and

trithorax group proteins

Giacomo CAVALLI. Montpellier, December 2006

CavalliCavalli LabLab

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

PcG and trxG proteins associate to multiple genomic loci

PH

DAPI

Polytene chromosome staining shows around 100 bands for each PcG protein

Merge

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

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

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

Histone H3 K27 methylation and Polycomb

Pc H3K27me3 Merge

There is a strong correlation between trimethylation of K27 (and K9) trimethylation and Polycomb recruitment at target loci.

Data from: Ringrose et al. (2004) Mol. Cell 16, 641

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

Features of PREs and TREs, and examples of how they are studied in Drosophila

(No PREs characterized yet in vertebrates)

1. Example of PcG-dependent spatial specific silencing of homeotic genes

PcG dependent derepression of a Ubx-lacZ reporter in embryonic territories where it is normally silenced

Silencing of a Ubx-lacZ reporter mimicking the wt behaviour of the Ubx gene, which is silenced in parasegments 1 to 5

bxd5.1 UbxlacZ reporter construct

Bxd 5.1 PRE Ubx prom LacZ mini-white

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.

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

3. Recruitment of PcG and trxG proteins to PREs: analysis in Fab-7 by a combination of immunostaining and FISH in polytene chromosomes (immuno-FISH)

24A

25E5

transgene

DAPI Immunostainingof PH protein

FISH Immuno-FISH

Fab-7 UAS-lacZ white

Transgene :

4. Recruitment of PcG and trxG proteins to PREs: analysis by ChIP

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

sd

en/inv

Bx-CHh

ci

Adh region

X

2L 2R

3L 3R

4

Montpellier tiling paths

Yale tiling paths

L82

X chromosome tip

bi (1)

(2)

mab2(3)

ct (4)

ph

gt/z

34D 35B 35D 36A2D 4C 5A 7B5D

elB

noc

stc

esg

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 20;4(6):e170

Example of a high-resolution description of PcG binding using ChIP and oligonucleotides arrays with one oligo every 100 bp

PH

PC

H3K27me3

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

5. Cross-talk between multiple copies of PREs

w w

w

Chromosome X

Pairing Sensitive Silencing: enhanced silencing of the mini-white reporter gene by multiple PRE copies

---> strong mini-white silencing---> weak silencing of the mini-white reporter gene

Transgenic Fab-7heterozygous

Transgenic Fab-7homozygous

PP mini-whiteFab-7

PcG-mediated silencing is enhanced by the presence of multiple copies of homologous PREs in the genome

Fab-X; Fab71

XX

III III

Fab-X

sd sd

BX-C BX-C

Fab-7 transgene

Endogenous Fab-7

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.

0

5

10

15

20

25

+ + -Endogenous Fab-7 at the BX-C (Chr.III)

3m

-

-

-

-

-

-Fab-X Fab-X;

Fab-71

WT

WT

Fab-X

Fab-X; Fab-71

+ +-Transgenic Fab-7 at sd (Chr.X)

Fab-7

Fab-7

Fab-7

Fab-7

sd

BX-C

sd

BX-C

sd

BX-C

Dapi Sd BX-C Merge

Homologous Fab-7 copies associate in the nucleus

% of interaction

Two identical Fab-7 can interact in the nucleus, leading to an increase of PcG-dependent silencing

Nucleus in a Rabl configuration

Giacomo.Cavalli@igh.cnrs.fr

For more info…

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

http://www.epigenome-noe.net

In french…

Inserm Actualités N. 201, Octobre 2006 (http://www.inserm-actualites.fr/index.php?id=562)