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UMR 599 Centre de Recherche en Cancérologie de Marseille

UMR 599 Centre de Recherche en Cancérologie de Marseille. L'empreinte phylogénétique : Identification de séquences régulatrices de la transcription chez les eucaryotes supérieurs È cole-chercheur INRA La Phylogénomique : Une aide à l’étude des grandes fonctions du vivant

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UMR 599 Centre de Recherche en Cancérologie de Marseille

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  1. UMR 599 Centre de Recherche en Cancérologie de Marseille L'empreinte phylogénétique : Identification de séquences régulatrices de la transcription chez les eucaryotes supérieurs Ècole-chercheur INRA La Phylogénomique : Une aide à l’étude des grandes fonctions du vivant Carry-le–Rouet – 11 au 14 décembre 2006 Jean Imbert Équipe Régulations Transcriptionnelles For a European Research Initiative: http://fer.apinc.org/

  2. From Bing Ren, Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, UCSD

  3. Genome-wide comparison of transcriptional activator families in eukaryotes C2H2 zinc fingers are found in 2% of all human genes, and they are by far the most abundant class of DNA-binding domains found in human transcription factors. From Tupler R, Perini G, Green MR. Nature 409: 832-833, 2001.

  4. CORE PROMOTER MODEL OF TYPICAL GENE PROMOTER AND REGULATORY REGIONS + 1 Enhancer Regulatory Elements TATA Box Inr Enhancer -4000 -500 -40 +50 +2000 Core Promoter Regulatory Promoter

  5. CTCF Modified from Jones and Kadonaga, Genes Dev. 14:1992-1996, 2000.

  6. TOOLS FOR THE IDENTIFICATION OF REGULATORY SEQUENCES AND THEIR COGNATE TRANSCRIPTION FACTORS 1. Identification and characterization of regulatory sequences - Gene reporter assays: transient or stable transfection (CAT, Luciférase, SEAP, GFP, b-gal, etc.) - In vitro transcription assay - Sequencing, database mining (web: TESS, Euk. Pr. Database, TRANSFAC, MATINSPECTOR, TFSEARCH, etc.) - Animal or cellular models: enhancer trap, enhancer knock-in, minichromosomes, etc. 2. Identification and characterization of specific transcription factors (TFs) - EMSA and sequels: UV-crosslinking, pull-down assay using biotynilated oligonucleotide - Footprint detection: * in vitro : nucleases (DNAse I hypersensitivity, S1 nuclease, Mmase) or chemical compounds * in vivo : genomic footprinting, Chromatin ImmunoPrecipitation (ChIP) and sequels 3. TFs physical and functional interactions - Transfection and biochemistry - ChIP-on-chip

  7. Promoter Regulatory Elements:Features and Facts • Degenerate sequence motifs • Length: 6 to 20 bp • Low complexity (8-12 bits) • Binding sites of transcription factors • Excess of binding sites over binding proteins in the nucleus • Most in vitro binding sites not functional in vivo • Some in vivo binding sites also not functional • Regulatory potentials depends on cooperative effects between multiple elements

  8. CpG islands • CpG dinucleotides are present at 20% of predicted frequency • CpG islands: >200 bp long, >50 %G+C, CpG >0.6 predicted • CpG islands account for 1% of the genome • 29,000 CpG islands are predicted in the human genome • ~60% of known genes have a CpG island near 5’ end • CpG island microarrays are promoter- and regulatory region-enriched arrays Weinmann et al. (2002) Genes & Dev. 16:235-244 Oberley et al. (2004) Methods Enzymol. 376:315-334

  9. From Bing Ren, Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, UCSD

  10. From Bing Ren, Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, UCSD

  11. TGF-b B7 Ag Ag/TCR/CD3 Signal 1 IL-2 B7/CD28 Signal 2 TGF-b/TGF-bR Other signal IL-2/IL2-R Signal 3 AP-1 AP-1 Stat5a,b SRF Stat5a,b NF-kB SATB1? Elf-1 Ets-1/2 AP-1 IL-2 Ra mRNA Exon 1 GATA-1-like CREB/ATF HMG-I(Y) NFAT AP-1 NFAT HMG-I(Y) Smad3 CREB/ATF HMG-I(Y) PRRVI [-8689,-8483] CD28rE PRRV [-7664,-7566] PRRIII [-3780,-3703] IL-2rE PRRI [-276,-244] PRRII [-137,-64] PRRIV [+3389,+3596] IL-2rE SBS [+7822,+8199] APC MHC I I I II II CD4+ T cell

  12. TPA BN000945

  13. Phylogenetic footprinting: two species comparison

  14. Exons: 1 2 3 4 5 6 7 k Regulatory Regions: VI V III I+II IV Homo Sapiens/Mus Musculus IL-2Ra locus dotplot comparison Homo Sapiens IL-2Ra 7 6 5 4 3 2 intron A Mus Musculus IL-2Ra gap SBS (SABT1)? SBS IV 1 I III V Schwartz, S., Zhang, Z., Frazer, K.A., Smit, A., Riemer, C., Bouck, J., Gibbs, R., Hardison, R., and Miller, W. (2000). PipMaker: A Web Server for Aligning Two Genomic DNA Sequences. Genome Res. 10, 577-586. update JI 17/10/05

  15. PRRIII Homo Sapiens EBSp GASp/GATA GASd/EBSd TTTCTTCTAGGAAGTACCAAACATTTCTGATAATAGAATTGAGCAATTTCCTGAT IIIIIIII--IIIIIIIII-IIIIIIIIIIIII-III-IIIIIIII-IIIIIIII TTTCTTCTGAGAAGTACCAGACATTTCTGATAAGAGAGTTGAGCAACTTCCTGAT - 3772 - 3718 - 1369 - 1315 Site I Site II Site III Mus Musculus IL-2rE

  16. - - - + + + FcgRI-GAS Site I GASd/EBSd IL-2 C3 C3 C2 C2 C1 1 2 3 4 5 6 Inducibles complex C2 and C3 are GAS-specific Constitutive complex C1 is EBS-specific GASd CD25/IL-2Ra GASd/EBSd EMSA probe: TTTCTTCTAGGAAGTACC AAAGAAGATCCTTCATGG EBSd Mouse site ITTTCTTCTGAGAAGTACC AAAGAAGACTCTTCATGG

  17. PRIMARY ACTIVATION XbaI XbaI + 1 TATA PRRI PRRIII NF-kB SRF 250XbaI -300 -3700 -3800 -100 -200 Elf-1 PRRII pTK CAT INDUCTION FOLD IL-2 pTK4 / 250XbaI 4.2 pTK4.CAT 1.33

  18. - 3769 CGTATACGTAATGCGCATATGCTA GASd EBSd - 3757 In vivo modification of the GASd/EBSd motif occupancy in response to CD2+CD28 costimulation in purified human primary T cells Primary T lymphocytes CD2+CD28 In Vitro 48h 24h ns - 3819 GASp/GATA EBSp - 3700 1 2 3 4

  19. The GASd/EBSd motif is the only putative regulatory element within PRRIII modified in vivo in response to an IL-2-dependent induction in human T lymphocytes INDUCIBLE TTTCTTCTAGGAAGTACCAAACATTTCTGATAATAGAATTGAGCAATTTCCTGAT AAAGAAGATCCTTCATGGTTTGTAAAGACTATTATCTTAACTCGTTAAAGGACTA - 3772 - 3718 GASd/EBSd GASp/GATA EBSp CONSTITUTIVE Lecine, P., Algarte, M., Rameil, P., Beadling, C., Bucher, P., Nabholz, M. and Imbert, J. Elf-1 and Stat5 bind to critical element in a new enhancer of the human interleukin-2 receptor alpha gene. Mol.Cell.Biol. 16: 6829-6840; 1996.

  20. Chromatin immunoprecipitation (ChIP) Formaldehyde cross-linking Sonocation to shear chromatin Specific Anti-bodies Immunoprecipitation Unbound fraction Bound fraction Reversing cross-linking Semi-quantitative PCR Assays

  21. IL-2 16h 3h M 1 2 3 4 5 6 7 8 9 input no Ab input no Ab Stat5b Stat5b Ets-1/2 Ets-1/2 p-Stat5b Stat5b and Ets-1/2 bind to IL-2Ra within human IL-2rE in vivo PRRIII ChIP primer design 1 TTCTGCCCTTAGCTTCTACCCCTCTCTACTTCTGGTTAACTATGGACCACACTCTGCTTC BZ3-1 BZ3-2 61 CTCAGGAACCACCTACCAAGGCCGTATCCATCCTTCAAGGACAATACGTGGGCCTTTCCT >>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>> 121 GATCACATCAGCTCAACAACTTTTCCCTCCTACATTTCAATTGCTCTTCTTACCATAATC >>> 181 ATTAGTATTCACCCCACTGTACGTCTAGAAAGAAAGTGGTCTTAAACCTAAGGGAAGGCA 241 GTCTAGGTCAGAAATTTGTTGTCCGCTGTTCTGAGCAGTTTCTTCTAGGAAGTACCAAAC BZ3-3 301 ATTTCTGATAATAGAATTGAGCAATTTCCTGATGAAGTGAGACTCAGCTTGCACTGTTGA <<<<<<<<<<<<<<<<<< 361 CCGGCTGTCCTGGATGAACCTAGTTACTTTTAACCAAATGTTCCTTTCTTGAACTTGTTC << 421 CTTTCTTGAACTTAATCTATC OLIGO start len tm gc% 3' seq BZ3-1 62 20 59.96 55.00 TCAGGAACCACCTACCAAGG BZ3-3 362 20 59.62 55.00 GGTCAACAGTGCAAGCTGAG BZ3-2 104 20 60.71 50.00 ATACGTGGGCCTTTCCTGAT

  22. A. Homo Sapiens/Mus musculus CD25/IL-2Ra gene

  23. A Well-Conserved but not Functional Candidate for a CD28 Responsive Enhancer

  24. The crucial CRE/TRE motif within CD25/IL-2RaPRRV/CD28rE is contained in a SINE/MIR repeat and is not conserved between Homo Sapiens and Mus Musculus Local identities (aligment n°1) 211-245 <--> 312-346 74% (35 nt) 261-312 <--> 347-398 62% (52 nt) 313-322 <--> 400-409 70% (10 nt) 323-371 <--> 414-462 61% (49 nt) 374-388 <--> 463-477 60% (15 nt) 389-444 <--> 480-535 75% (56 nt) 447-482 <--> 536-571 53% (36 nt) 491-542 <--> 572-623 67% (52 nt) 544-602 <--> 624-682 61% (59 nt) 603-664 <--> 684-745 66% (62 nt) 666-686 <--> 746-766 57% (21 nt) 690-709 <--> 767-786 85% (20 nt) 712-744 <--> 787-819 58% (33 nt)

  25. Functional annotation of Homo sapiens ERBB2 gene: from text mining to motif discovery using phylogenetic footprinting

  26. distal promoter proximal promoter NM_001005862 • 42 introns • 17 different transcripts by alternative splicing, all with introns • 18 putative different protein isoforms • 11 probable alternative promoters • 7 non overlapping alternative last exons • 11 probable alternative promoters • 2 RefSeq mRNAs: NM_00448 and NM_001005862 at least 2 promoters The ERBB2 gene in Homo sapiens According to AceView: http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/

  27. ERBB2 locus Homo sapiens Mus musculus CpG CpG CpG CpG PERLD1 ERBB2b, variant 2 mRNA, 30 exons C17orf37 ERBB2a, variant 1 mRNA, 27 exons

  28. Phylogenetic footprinting: multi-species comparison

  29. Species choice for phylogenetic footprinting analysis Homo sapiens Pan troglodytes Rhesus maccaca Rattus norvegicus ESPERR UCSC Mus musculus Bos taurus Canis familiaris Monodelphis domestica

  30. Homo sapiens ERBB2 locus chr17:35,092,919-35,143,441 UCSC Genome Bioinformatics: http://genome.ucsc.edu/ ESPERR Regulatory Potential (7 species): alignments of human, chimpanzee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), dog (canFam2), and cow (bosTau2).

  31. VISTA Tools: mVISTA and rVISTAhttp://genome.lbl.gov/vista/ Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 2004 Jul 1;32 (Web Server issue):W273-9

  32. mVISTA • Limits: • sequence numbers: 2-100 • size: ? Mayor C., Brudno M., Schwartz J. R., Poliakov A., Rubin E. M., Frazer K. A., Pachter L. S. and Dubchak I. (2000) VISTA: Visualizing Global DNA Sequence Alignments of Arbitrary Length. Bioinformatics, 16:1046.

  33. ERBB2 locus

  34. rVISTA • Limits: • sequence numbers: 2-100 • size: 20 Kb per sequence for a total of 60 Kb Loots, G., Ovcharenko,I., Pachter,L., Dubchak,I., Rubin, E. rVISTA for comparative sequence-based discovery of functional transcription factor binding sites. (2002) Genome. Res. 12:832-839

  35. Hs versus Md ERBB2a regulatory regions (13,883 bases)

  36. GATA1 Input no Ab CR3 CR5 CR7 CR8 1 2 3 4 5 6 7 Chromatin Immunoprecipitation on ERBB2 Gene Putative Regulatory Regions in Human Breast Cancer Cell Line BT474 GATA3 GATA4 GATA6 GATA2 M: Marker; 1,8: Input; 2,9: NoAb; 3,11: GATA1;;4,11: GATA2; 5,12: GATA3; 6,13: GATA4 ; 7,14: GATA6

  37. Parfois on peut aller très vite de l’ordinateur à la paillasse…

  38. Selection of CD19 B-cell specific regulatory sequence and design of CD19-GFP lentiviral vector Moreau, T., F. Bardin, J. Imbert, C. Chabannon, and C. Tonnelle. 2004. Restriction of transgene expression to the B-lymphoid progeny of human lentivirally transduced CD34+ cells. Mol.Ther,. 2004, in press.

  39. 1529 CR1 StuI kB kB GATA PvuII GATA Myb PstI GATA repeat Bright SINE/Alu HinfI Pax5 GATA repeat gap CR2 AvaI 1 1 1274 2xkB kB E-box Myb kB EcoRI SspI XbaI BglI Pax Myb Ikaros Ikaros Pax Bright E-box GATA Pax EBF Pax GA_rich SINE/Alu SINE/MIR SINE/Alu LINE/L2 proximal distal Computational analysis of homologies between Human and Mouse CD19 gene 5’ regions

  40. A. Recombinant CD19-GFP lentiviral vectors B. GFP expression in the progeny of transduced CD34+ progenitor cells differentiated in vitro B cells Myeloid cells NK cells Herythroid cells From Moreau et al., Mol.Ther, 2004.

  41. Genomatix http://www.genomatix.de/ DiAlign TF: Multiple alignment plus TF sites

  42. Galaxy at Penn State University Center for Comparative Genomics and Bioinformatics • Galaxy features connections to UCSC Table Browser, EnsMart, and contains hundreds of tools. • Some examples: • Extraction of multiple alignments corresponding to a genomic region, • Finding exons overlapping SNPs, compute phastCons scores for a set of genomic ranges, • Building histograms, compute correlations, draw scatterplots, search using simple conditional • statements • And much much more... Galaxy website http://www.bx.psu.edu/cgi-bin/trac.cgi

  43. IL2RA

  44. KEGG: hsa04630

  45. ORegAnno Open Regulatory Annotation: http://www.oreganno.org PIPMaker: http://pipmaker.bx.psu.edu/pipmaker/ UCSC Genome Browser: http://genome.ucsc.edu PromoSer The mammalian promoter service http://biowulf.bu.edu/zlab/PromoSer VISTA Tools: mVISTA and rVISTAhttp://genome.lbl.gov/vista/ GenomatixTM: http://www.genomatix.de Galaxy website: http://www.bx.psu.edu/cgi-bin/trac.cgi Evolutionary Conserved Region (ECR) Browser: ECR Browser http://ecrbrowser.dcode.org/ IdConvert: http://idconverter.bioinfo.cnio.es/

  46. Immunology Center of Marseille-Luminy Salvatore Spicuglia Sanjeev Kumar Pierre Ferrier INSERM U363, Paris Fabrice Gouilleux Curie Institute, Orsay Jacques Ghysdael ISREC, Lausanne Philip Bücher Markus Nabholz ICRF, London Carol Beadling Doreen Cantrell Genzentrum, Martinsried Patrick Baeuerle TAGC, INSERM EMI206, Marseille Béatrice Loriod Benoît Ballester Rémi Hougatte NHLBI/NIH Hyoung Pio Kim Warren Leonard INSERM UMR599 Cancer Institute of Marseille Director : Françoise Birg Transcription Team Michèle Algarté Pierre Cauchy Régis Costello Brigitte Kahn-Perlès Guoqiang Hua Patrick Lécine Carol Lipcey Pascal Rameil Jung-Hua Yeh Bing Zhu Jean Imbert Immunology Team Chantal Cerdan Jacques Nunès Daniel Olive Gene & Cellular Therapy Team Thomas Moreau Christian Chabannon Cécile Tonnelle Oncology Team José Adelaïde Daniel Birnbaum

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