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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/
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From Bing Ren, Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, UCSD
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From Tupler R, Perini G, Green MR. Nature 409: 832-833, 2001. 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.
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CORE PROMOTER MODEL OF TYPICAL GENE PROMOTER AND REGULATORY REGIONS EnhancerRegulatory Elements + 1 Inr EnhancerTATA Box Core Promoter Regulatory Promoter -40 +50-4000 -500+2000
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Modified from Jones and Kadonaga, Genes Dev. 14:1992-1996, 2000. CTCF
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cis-acting regulatory elements From Pennacchio, L. A. and E. M. Rubin. 2001. Genomic strategies to identify mammalian regulatory sequences. Nat. Rev. Genet. 2:100-109. Promoter: Sequence of DNA near the 5' end of a gene that acts as a binding site for RNA polymerase and from which transcription is initiated. Enhancer: Control element that elevates the levels of transcription from a promoter, independent of orientation or distance. Locus control region (LCR): Confers tissue-specific temporally regulated expression of linked genes. LCRs function independently of position, but they are copy number dependent and open the nucleosome structure so that other factors can bind. LCRs affect replication timing and origin usage. Boundary element/insulator: DNA sequence that prevents the activation or inactivation of transcription by blocking the effects of surrounding chromatin. Silencer: Control element that suppresses gene expression independent of orientation or distance. Matrix attachment region (MAR)/scaffold attachment region (SAR): DNA sequence that binds the nuclear scaffold and can affect transcription. These elements probably form higher- order looped structures within chromosomes and influence gene expression by separating chromosomes into regulatory domains.
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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, - 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
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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
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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 CpG islands Weinmann et al. (2002) Genes & Dev. 16:235-244 Oberley et al. (2004) Methods Enzymol. 376:315-334
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From Bing Ren, Laboratory of Gene Regulation, Ludwig Institute for Cancer Research, UCSD
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IL-2/IL2-R Signal 3 IL-2 I II I TGF- TGF- /TGF- R Other signal IL-2 R mRNA PRRII [-137,-64] PRRI [-276,-244] Elf-1 HMG-I(Y) PRRIV [+3389,+3596] IL-2rE PRRIII [-3780,-3703] IL-2rE Stat5a,b HMG-I(Y) Stat5a,b GATA-1-like Ets-1/2 PRRVI [-8689,-8483] CD28rE NF- B SRF CREB/ATF AP-1 HMG-I(Y) NFAT Exon 1 AP-1 NFAT SATB1? SBS [+7822,+8199] CREB/ATF AP-1 Smad3 PRRV [-7664,-7566] CD4 + T cell APC B7 B7/CD28 Signal 2 Ag/TCR/CD3 Signal 1 Ag MHC I
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TPA BN000945
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Phylogenetic footprinting: two species comparison
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Homo Sapiens/Mus Musculus IL-2R locus dotplot comparison 1234567Exons: SBS (SABT1)? k Homo Sapiens IL-2R III I 1 intron A IV SBS gap 2 3 4 5 6 7 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 IIIVII+IIIVRegulatory Regions:V Mus Musculus IL-2R V
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TTTCTTCTAGGAAGTACCAAACATTTCTGATAATAGAATTGAGCAATTTCCTGAT IIIIIIII--IIIIIIIII-IIIIIIIIIIIII-III-IIIIIIII-IIIIIIII TTTCTTCTGAGAAGTACCAGACATTTCTGATAAGAGAGTTGAGCAACTTCCTGAT Site ISite IISite III GASp/GATA GASd/EBSd EBSp PRRIII IL-2rE - 1369- 1315 - 3772- 3718 Homo Sapiens Mus Musculus
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IL-2 GASd/EBSd Site I Fc RI-GAS C2 C1 C3 C2 C3 123456 - + - + - + Inducibles complex C2 and C3 are GAS-specific Constitutive complex C1 is EBS-specific GASd CD25/IL-2R GASd/EBSd EMSA probe: TTTCTTCTAGGAAGTACC AAAGAAGATCCTTCATGG EBSd Mouse site I TTTCTTCTGAGAAGTACC AAAGAAGACTCTTCATGG
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250XbaI TATA -3700-3800 PRRII + 1 -100-200 PRRI -300 PRIMARY ACTIVATION XbaI NF- B SRF Elf-1 pTK CAT PRRIII IL-2 pTK4.CAT pTK4 / 250XbaI INDUCTION FOLD 4.2 1.33
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1234 ns In Vitro 24h 48h CD2+CD28 Primary T lymphocytes - 3700 - 3819 GASp/GATA EBSp CG TA TA CG TA AT GC GC AT AT GC TA GASd EBSd - 3769 - 3757 In vivo modification of the GASd/EBSd motif occupancy in response to CD2+CD28 costimulation in purified human primary T cells
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TTTCTTCTAGGAAGTACCAAACATTTCTGATAATAGAATTGAGCAATTTCCTGAT AAAGAAGATCCTTCATGGTTTGTAAAGACTATTATCTTAACTCGTTAAAGGACTA GASp/GATA GASd/EBSd EBSp - 3772- 3718 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 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.
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Formaldehyde cross-linking Sonocation to shear chromatin Specific Anti-bodies Immunoprecipitation Bound fraction Unbound fraction Reversing cross-linking Semi-quantitative PCR Assays Chromatin immunoprecipitation (ChIP)
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M 1 2 3 4 5 6 7 8 9 IL-2 16h 3h input no Ab Stat5b p-Stat5b Ets-1/2 Stat5b and Ets-1/2 bind to IL-2R 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
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A. Homo Sapiens/Mus musculus CD25/IL-2R gene
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A Well-Conserved but not Functional Candidate for a CD28 Responsive Enhancer
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The crucial CRE/TRE motif within CD25/IL-2R PRRV/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)
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Functional annotation of Homo sapiens ERBB2 gene: from text mining to motif discovery using phylogenetic footprinting
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The ERBB2 gene in Homo sapiens According to AceView: http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ 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 NM_001005862 proximal promoterdistal promoter
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ERBB2 locus ERBB2a, variant 1 mRNA, 27 exons ERBB2b, variant 2 mRNA, 30 exonsPERLD1C17orf37 CpG Homo sapiens Mus musculus
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Phylogenetic footprinting: multi-species comparison
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Species choice for phylogenetic footprinting analysis Homo sapiens Mus musculus Bos taurus Canis familiaris Monodelphis domestica Pan troglodytes Rhesus maccaca Rattus norvegicus ESPERR UCSC
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UCSC Genome Bioinformatics: http://genome.ucsc.edu/ Homo sapiens ERBB2 locus chr17:35,092,919-35,143,441 ESPERR Regulatory Potential (7 species): alignments of human, chimpanzee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), dog (canFam2), and cow (bosTau2).
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VISTA Tools: mVISTA and rVISTA http://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
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mVISTA 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. Limits: sequence numbers: 2-100 size: ?
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ERBB2 locus
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rVISTA 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 Limits: sequence numbers: 2-100 size: 20 Kb per sequence for a total of 60 Kb
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Hs versus Md ERBB2a regulatory regions (13,883 bases)
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Chromatin Immunoprecipitation on ERBB2 Gene Putative Regulatory Regions in Human Breast Cancer Cell Line BT474 GATA2 GATA3GATA6 1 2 3 4 5 6 7 CR5 Inputno Ab GATA1 CR7 CR8 CR3 M: Marker; 1,8: Input; 2,9: NoAb; 3,11: GATA1;;4,11: GATA2; 5,12: GATA3; 6,13: GATA4 ; 7,14: GATA6 GATA4
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Parfois on peut aller très vite de l’ordinateur à la paillasse…
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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.
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EcoRI SspIXbaIBglI distal PaxMyb 2xkB Ikaros kB IkarosPax E-box Bright Myb E-boxGATA GA_richSINE/AluSINE/MIRSINE/Alu LINE/L2 proximal PaxEBF kB Pax CR2 CR1 AvaI HinfI PstI PvuII StuI GATA Bright Pax5 repeat SINE/Alu repeat GATA Myb GATA kB gap 11274 1529 1 Computational analysis of homologies between Human and Mouse CD19 gene 5’ regions
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A. Recombinant CD19-GFP lentiviral vectors B. GFP expression in the progeny of transduced CD34 + progenitor cells differentiated in vitro From Moreau et al., Mol.Ther, 2004. B cells Myeloid cells NK cells Herythroid cells
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Genomatix http://www.genomatix.de / DiAlign TF: Multiple alignment plus TF sites
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Galaxy features connections to UCSC Table Browser, EnsMart, and contains hundreds of tools.UCSC Table BrowserEnsMart 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 Galaxy at Penn State University Center for Comparative Genomics and Bioinformatics
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IL2RA
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KEGG: hsa04630
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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 rVISTA http://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/
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INSERM UMR599 Cancer Institute of Marseille Director : Françoise Birg Immunology Team Chantal Cerdan Jacques Nunès Daniel Olive Gene & Cellular Therapy Team Thomas Moreau Christian Chabannon Cécile Tonnelle Oncology Team José Adelaïde Daniel Birnbaum 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 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
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