1. Cavalli Lab Introduction in: Epigenetic regulation by Polycomb and trithorax group proteins Giacomo CAVALLI. Montpellier, December 2006

2. Cavalli Lab 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

3. Where on the chromosomes are condensed and open chromatin located? Condensed chromatinOpen 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

4. 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

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

6. PcG and trxG proteins associate to multiple genomic loci PH DAPI Polytene chromosome staining shows around 100 bands for each PcG protein Merge

7. 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

8. protein motifs Members of the PcG and of the trxG Gene trxG protein motifsGene PcG 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-1SET (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) PhoRC Complex

9. OFF ON trxG Maintenance of active states (open chromatin) PRE Target 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

10. Histone H3 K27 methylation and Polycomb PcH3K27me3 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

11. 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

12. Features of PREs and TREs, and examples of how they are studied in Drosophila (No PREs characterized yet in vertebrates)

13. 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 PREUbx promLacZmini-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.

14. 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 -/+ Fab-7UAS-lacZwhite

15. 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 Immunostaining of PH protein FISH Immuno-FISH Fab-7UAS-lacZwhite Transgene :

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

17. 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

18. 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 34D35B35D36A2D4C5A7B5D 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

19. Example of a high-resolution description of PcG binding using ChIP and oligonucleotides arrays with one oligo every 100 bp PH PC H3K27me3

20. 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 members occupied 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) PRC2PRC1 66 94 300 986 164 55 365 12131 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

21. 5. Cross-talk between multiple copies of PREs

22. 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-7 heterozygous Transgenic Fab-7 homozygous P P mini-white Fab-7

23. PcG-mediated silencing is enhanced by the presence of multiple copies of homologous PREs in the genome Fab-X; Fab7 1 X X III Fab-X sd BX-C Fab-7 transgene Endogenous Fab-7 deletion, named Fab-7 1 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.

24. 0 5 10 15 20 25 ++ - Endogenous Fab-7 at the BX-C (Chr.III) 3m3m - - - - - - Fab-XFab-X; Fab-7 1 WT Fab-X Fab-X; Fab-7 1 ++ - Transgenic Fab-7 at sd (Chr.X) Fab-7 sd BX-C sd BX-C sd BX-C DapiSdBX-CMerge Homologous Fab-7 copies associate in the nucleus % of interaction

25. Two identical Fab-7 can interact in the nucleus, leading to an increase of PcG-dependent silencing Nucleus in a Rabl configuration

26. 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)