In vivo protein-DNA interactions

1. In vivo protein-DNA interactions Giacomo Cavalli Institut de Génétique Humaine, CNRSMontpellier, France UE Méthodologie, 11 April, 2014

2. Compaction by higher order determinants 10,000 nm DNA compactioninthenucleus 11 nm 30nm 1bp (0.3nm) Compaction of DNA by histones

3. Implication of PcG proteins in dynamic gene regulation Cellular Memory System Differentiation Cell fate determination Proliferation Cell cycle Control HOX genes PcG proteins Signaling genes Cell cycle genes TFs of developmental networks Cancer development Stem cell plasticity Developmental pathways

4. Schematic mechanism of Polycomb mediated silencing Histone Methyl Transferase Me3 K27 Me3 K27 Core PRC2 E(z) H3 H3 H3 H3 Esc Su(z)12 Target gene Nurf-55 PRE

5. Psc Scm Ph dRing Pc Schematic mechanism of Polycomb mediated silencing Histone Methyl Transferase Me3 K27 Me3 K27 Core PRC2 E(z) H3 H3 H3 H3 Esc Su(z)12 Target gene Nurf-55 PRE Core PRC1 Me3 K27 Me3 K27 H3 H3 H3 H3

6. Psc Scm Ph dRing Pc Schematic mechanism of Polycomb mediated silencing Histone Methyl Transferase Me3 K27 Me3 K27 Core PRC2 E(z) H3 H3 H3 H3 Esc Su(z)12 Target gene Nurf-55 PRE Ub E3ligase Core PRC1 Me3 K27 Me3 K27 Ub K119 Ub K119 H3 H3 H3 H3

7. Psc Scm Ph dRing Pc Schematic mechanism of Polycomb mediated silencing Histone Methyl Transferase Me3 K27 Me3 K27 Core PRC2 E(z) H3 H3 H3 H3 Esc Su(z)12 Target gene Nurf-55 PRE Ub E3ligase Core PRC1 Me3 K27 Me3 K27 Ub K119 Ub K119 H3 H3 H3 H3 H2A H2A H2A H2A ATP-dependent chromatin remodeling

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

9. Genome-wide identification of downstream PcG target genes « ChIP-on-chip » approach

10. The ChIP on chip approach ChIP DNA chip Cross-link chromatin 1st generation microarrays Produce 2 KB PCR fragments of overlapping genomic DNA fragments Produce soluble chromatin 2nd generation microarrays whole genome coverage with 1,000,000 long oligonucleotides, i.e. 1 Oligo per 120 bp of euchromatin IP step Produce fluorescent labeled probes Protein IP Obtain the profile Hybridize to the DNA chip Control IP

11. 200Kb Dynamicfunction of Polycombproteins and cellproliferation Embryos -Schuettengruber et al 2009 H3K27me3 PC Ph S2 cells data-Schwartz et al 2006 H3K27me3 PC Psc http://www.purl.org/NET/polycomb

12. ChIP on chip validation: Comparing ChIP on chip data with a chromatin profiling using an independent technology called DamID In DamID, the chromatin protein of interest is fused to the bacterial Dam-methylase and the construct is transfected into the cells of interest. The protein of interest drives the Dam partner to its targets, and the methylase puts a methyl mark at the “A” of GATC sequences. Methylated DNA is then isolated and hybridized onto microarrays of interest

13. Correspondence between ChIP on chip and DamID data

14. toy / PAX6 ey / PAX6 26.4% Maternal genes eyg / PAX6(5A) 73.6% 23.1% N=53 eya / EYA1-4 so / SIX1/2 76.9% shf / WIF1 Optix / SIX3/6 dac / DACH1-2 Gap genes N=13 Eye specification 27.3% Pair-rule genes 72.7% N=11 40.7% Segment polarity genes 59.3% N=54 Homeotic genes 100 % N=8 No target 52.4% Direct Hox gene targets 47.6% PcG target N=21 Signaling pathways interacting with RDGN genes: PcG target genes regulate genes at multiple layers of transcriptional cascades FLYMOUSE HUMAN toy2 Pax6 PAX6 ey1 Pax6 PAX6 eyg (toe)1 - - Optix1 Six6 SIX6 shfWif1 WIF1 eya2Eya1-4EYA1-4 so1 Six1 SIX1 dac1 Dach1 DACH1 hh1Shh SHH dpp1Bmp2 BMP2 Additional factors involved in eye development: oc1 Otx1 OTX1 ato3 Atoh1-8 ATOH1-8 tsh2- - bi1 Tbx2 TBX2

15. The evolution of ChIP: massive sequencing of the immunoprecipitated chromatin DNA ChIP-Seq Library construction H3K36 ChIP 100bp 1Kb+ ~5-10ng Polish ends 5’ 3’ Taq extend 600bp 500bp A A 400bp Ligate Solexa Linkers 300bp 200bp 100bp

16. Laser Sequence one base at a time T A C G Illumina sequencing Linker ligated DNA Amplify to form clusters

17. Flow cell imaging by microscopy 60 X objective: thousands tiff images / hundred thousands of images per run.

18. Chromatin Immunoprecipitation Tag Sequencing After obtaining the sequences, they are positioned on the genome by automated algorythms (like Blast but quicker) and each tag is thus assigned its position on the genome. These profiles can then be quantified and analyzed just like normal ChIP on chip profiles

19. Identification of new PcG target genes • PcGtargets (PC/PH/H3K27me3) 305 maintained New domain PH 0 181 embryos PC 0 145 H3K27me3 0 350 PH eye discs 0 275 PC 0 353 H3K27me3 0 fd96Ca fd96Cb danr dan (+) (-) Anna Delest

20. PRE position is highly conserved in Drosophila species D.Melanogster vs D.Yakuba PH Mel PH Yak PC Mel PC Yak K27 Mel K27 Yak PHO Mel PHO Yak DSP1 Mel DSP1 Yak K4 Mel K4 Yak Wnt4 wg → species-specific differences can be used to study PRE sequence features Bernd Schüttengruber

21. Exploiting In vivo protein-DNA interactions to learn about the three dimensional conformation of chromatin

22. PREs are sometimes located at positions overlapping the proximal gene promoter, but in other instances they can be at tens of kilobases away from it. How can PcG proteins repress transcription in all these cases? 28 kb > 30 kb Mecanisms ? PRE PcG proteins

23. "LOOPING" "SPREADING" PRE PRE PcG proteins PcG proteins "SPREADING versus LOOPING" Two models have been proposed in order to explain how PcG proteins repress their target genes: 1.They might spread from the PRE into flanking chromatin, covering the whole domain including the target promoter 2.Alternatively, they might reach the promoter via direct looping of the PRE and establishment of protein-protein contacts. Interestingly, at some endogenous target genes PREs are located at very large distance from the promoter and they are flanked by elements called: "chromatin insulators"

24. Insulators • Insulators are divided into three classes depending on their abilities Enhancer blockers En. Ins. En. Gene Chromatin boundaries Ins. En. Gene Ins. Insulators that can be "bypassed" En. Ins. Ins. Gene • One insulator can have many of these properties

25. The gypsy insulator ● DNA element isolated from the drosophila gypsy retrotransposon ● This sequence contains 12 binding sites for the Su(Hw) protein, that is required for insulator function En. Ins. Ins. Gene Model of nuclear chromosomal architecture based on insulators interaction Insulator bypass model Domain A Domain B Gerasimova et al, Mol. Cell, 2000 Insulating proteins

26. Insulator Insulator PRE PRE red red DPRE DPRE brown brown DIns. DIns. orange orange yellow yellow white white Bypass of the gypsy insulator by a PRE     yellow white yellow white Insulator Insulator Expression of white Expression of white Yes! the PRE can bypass 2 insulators Enhancer PRE Gene Gene Insulator Insulator

27. 1kb 1kb 35 30 ●PcG proteins are able to spread from a PRE into a neighboring region of several kb. This spreading is blocked by one insulator 25 20 15 10 yellow white 5 Insulator Insulator 0 PRE 0 ●Two insulators build a chromatin domain fully shielded from invasion by PcG proteins 5 10 15 20 25 30 35 ChIP analyisis of the molecular landmarks of insulator bypass PC pupal stage ●PcG proteins bound to the PRE can reach a downstream promoter without coating an insulated chromatin domain Fold enrichment Fold enrichment PH pupal stage

28. The data shown before provide good evidence for a spreading process Can we get direct evidence for looping?

29. 5 DNA purification and quantitative PCR analysis 4 2 3 Ligation Extensive dilution Chromatin digestion Chromosome Conformation Capture (3C) technology: 3C technology allows to convert chromosomal interaction events into DNA ligation events that can be analyzed by PCR Biological material 1 Main steps of 3C technology Formaldehyde-fixed nuclei preparation

30. -15kb -10kb -5kb 0 +5kb +10kb +15kb 0.5% 0.4% 0.3% PRE Prox. Ins. 0.2% 0.1% 0.0% Two gypsy insulators build a chromatin loop (P)(S)YSW-22E lines - Adult H3C - distal gypsy insulator anchor Interaction level in percentage of input CG33543-RC Nplp4-RA yellow Dist. Ins. mini-white CG4238-RF (P)(S)YSW transposon CG15353-RA tRNA:CR31940-RA tRNA:CR31943-RA tRNA:CR31669-RA tRNA:CR31939-RA tRNA:CR31944-RA Anchor

31. In summary, both spreading and looping models could be correct, each one accounting for a particular context yellow yellow mini-white yellow yellow mini-white PRE PRE b w br b w br Dist. Ins. Prox. Ins. Dist. Ins. PcG proteins PRE close to its target promoter PRE distant from its target promoter "SPREADING" "LOOPING" + PRE PRE Comet et al, Dev. Cell 2006

32.  High-resolution 3C is appropriate to study chromatin conformation How PcG proteins and insulators might work in the cell nucleus Nucleus PcG bodies Insulator bodies Insulator-binding protein complexes

33. Analysis of Hox gene contacts by 4C • We developed a new 4C method based on “biotinylated primer extension” streptavidin bead Biotinylated Primer GGGGG CCCCC • The amplified material is then hybridized to a Microarray (Roche Nimblegen) • We used the Fab-7 PRE sequence as a bait, which negatively regulates the Abd-B gene in the BX-C Itys COMET

34. 20 20 18 18 16 16 14 14 12 12 10 10 8 8 6 6 4 4 2 2 0 0 G G G G G 1kb 500bp 400bp G G G G G 300bp 200bp 100bp Modification and control of the 4C procedure 3C preparation 2 Ins. / 1 Ins. 2 Ins. / 1 Ins. Unknown partner Anchor fragment 1. Copies number ratio Copies number ratio Biotinylated-primer extension 3C 4C 4C 4C BEFORE amplification BEFORE amplification AFTER amplification INPUT 2. Affinity purification on streptavidin beads 3. “In situ” linker synthesis Unknown partners ligated to the anchor fragment 4. Quantitative amplification by real-time PCR Anchor fragment 5. Genomic DNA-Chip Hybridization Primer dimers Denaturing 4% agarose gel

35. probabilities at scale = 3 probes probabilities at scale = 1 probe 4C data analysis by generation of domainograms Normalized profile intensities for each probe iare transformed into rank based scores Qi, which are combined into Siw multiscale scores and transformed as Piw probabilities using Fisher's Chi square law. Piw at scale = N probes The Piw values represent probabilities of 4C events as a function of chromatin domain size Legend: N is the total number of probes ri is the rank of probe i Piw in false color Piw at Log scale = N probes Benjamin LEBLANC

36. 10-5000 10-500 10-50 10-10 10-1 Major Fab-7 4C hits are Polycomb bound regions 4C Domainogram 3R 1Mb 3R 1Mb Fab-7 ANT-C prospero E5-ems NK-C grn hth pnt Drop BX-C srp-pnr ss Polycomb ChIP Domainogram

37. Simplified Hi-C procedure • Fix nuclei of 16-18 hr embryos • Digestion with 4-cutter DpnII • Ligation and DNA purification as 3C • Sonication and selection of ~800 bp • Deep paired-end sequencing

38. Hi-C efficiently reproduces known 3C contacts

39. Chromatin contact features Matrix diagonal is not homogeneous

40. Polycomb-mediated interactions Bantignies et al., 2011

41. http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.htmlhttp://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html References: Schüttengruber et al. (2009) PLoS Biol 7(1): e1000013; Comet et al. Dev Cell11, 117-124 and PNAS , 108(6):2294-9; Bantignies et al. Cell144, 214-26, Sexton et al. Cell 148, 458-472 Bernd SCHÜTTENGRUBER Nicolas NEGRE Benjamin LEBLANC Anna DELEST Itys COMET Tom SEXTON ERC EU - 7FP CNRS, ARC French ministry of research