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Introduction in: Epigenetic regulation by Polycomb and trithorax group proteins

Introduction in: Epigenetic regulation by Polycomb and trithorax group proteins. Giacomo CAVALLI. Montpellier, December 2006. The chromatin Yin / Yang. Heterochromatin. Euchrochromatin. - Heavily condensed - Gene poor Silent genes Late replicating DNA hypermethylated

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Introduction in: Epigenetic regulation by Polycomb and trithorax group proteins

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

  2. 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

  3. 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

  4. 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 Polycomb (PcG) and trithorax (trxG) group proteins: epigenetic regulators of genome function

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

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

  7. 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

  8. Members of the PcG and of the trxG PcG Gene protein motifs 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 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 bromo domain Brm complex brahma (brm) (DNA dependent ATPase/helicase)

  9. 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

  10. 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.

  11. 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

  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 bxd5.1 UbxlacZ reporter construct Bxd 5.1PRE Ubxprom 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.

  14. Fab-7 UAS-lacZ white 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 +/+

  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) Fab-7 UAS-lacZ white Transgene : transgene 24A 25E5 DAPI FISH Immuno-FISH Immunostaining of PH protein

  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) Cross-link cells or embryos with formaldehyde to induce protein-DNA crosslinks 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 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. Montpellier tiling paths Yale tiling paths X sd X chromosome tip 2R 2L Adh region en/inv 3R 3L L82 Hh Bx-C 4 ci bi (1) esg noc ph mab2(3) stc elB gt/z ct (4) (2) 2D 4C 5A 5D 7B 34D 35B 35D 36A 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 H3K27me3 PC PH

  20. 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 66 365 MEIS/EVX 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 Boyer et al. Nature. 2006 May 18;441(7091):349-53. Epub 2006 Apr 19 Lee et al. Cell. 2006 Apr 21;125(2):301-13

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

  22. Fab-7 mini-white P P Pairing Sensitive Silencing: enhanced silencing of the mini-white reporter gene by multiple PRE copies Chromosome X TransgenicFab-7 heterozygous TransgenicFab-7 homozygous w w w ---> weak silencing of themini-white reporter gene ---> strongmini-white silencing

  23. PcG-mediated silencing is enhanced by the presence of multiple copies of homologous PREs in the genome Fab-X Fab-X; Fab71 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.

  24. Homologous Fab-7 copies associate in the nucleus Dapi Sd BX-C Merge WT sd % of interaction - 25 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 WT Fab-X Fab-X; Fab-71 3mm - Transgenic Fab-7 at sd (Chr.X) + + + + - Endogenous Fab-7 at the BX-C (Chr.III)

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

  26. 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) Giacomo.Cavalli@igh.cnrs.fr

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