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Paulo Arruda 02-06-2011

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  1. BG - 581 COMO DESCOBRIR OS GENES REGULADOS POR UM FATOR DE TRANSCRIÇÃO CHROMATIN IMMUNOPRECIPITATION (CHIP) SEQUENCING Paulo Arruda 02-06-2011

  2. O DANA GENOMICO ESTÁ SEMPRE ASSOCIADO A PROTEÍNAS Uma das funçõesdesseconjunto de proteínas é manteros genes inativos

  3. AS PROTEINAS ASSOCIADAS AO DNA GENOMICO FORMAM COMPLEXOS CONTENDO CO-REPRESSORES E ENZIMAS

  4. A ATIVAÇÃO DE UM GENE REQUER A DISSOCIAÇÃO DOS COMPLEXOS CO-REPRESSORES/ENZIMAS E A LIGAÇÃO DOS FATORES DE TRANSCRIÇÃO

  5. A ATIVAÇÃO DA TRANSCRIÇÃO ENVOLVE UM CONJUNTO GRANDE DE DIFERENTES PROTEÍNAS SENDO A ESPECIFICIDADE DETERMINADA PELOS FATORES DE TRANSCRIÇÃO

  6. COMPONENTES DA ORQUESTRA

  7. COMO SERÁ QUE MILHARES DE ORQUESTRAS JUNTAM SEUS COMPONENTES AO MESMO TEMPO? (A) Cooperative chromatin remodeling events (left) induced by ATP-dependent factors (purple) and histonemodifyingacetyltransferase activities (red and orange) through their interactions (broken arrows) with each other and some transcriptional regulators (blue circle) that have access (thick arrow) to specific DNA sequences through gene-specific nucleosome positioning effects. (B) Chromatin remodeling lead to nucleosome shifting relative to specific target sequences as well as histoneacetylation (grey lines), which together likely allow full template accessibility to other transcription factors (blue diamond and Sp1, green hexagon) and the core machinery (right). Activators and their co-regulators can interact (thick black arrow) with multiple components of the core initiation machinery; (C) Activated transcription (black arrow) requires the assembly of a large oligomeric initiation complex and, likely, multiple concerted signals (red arrows) from several gene regulators. Pre-assembly model for transcription initiation. A holoenzyme (i.e., a complex containing chromatin remodeling factors, multiple co-regulators, RNA polymerase, the core initiation machinery, and RNAprocessing factors) is recruited via cooperative interactions with several gene regulators.

  8. OS NUCLEOSSOMOS REPRESENTAM O MENOR NÍVEL DO COMPLEXO DNA/PROTEÍNA QUE ESTRUTURA A CROMATINA

  9. O COMPLEXO DNA/PROTEÍNA PODE SER FIXADO • Tratamento com formaldeidofaz o “cross-link” das moleculas de proteinasao DNA “congelandoporexemploosFatores de Transcrição no seusítio de ligaçãoao DNA • Cross-linking pode ser feitoem: • Celulasemsuspensão • Cultura de célulasemplaca • Tecidos • Cortes histológicos

  10. O ISOLAMENTO DO DNA GENOMICO CARREGA AS ROTEINAS ASSOCIADAS Apóssonicaçãosãoproduzidosfragmentos de DNA de ~ 500 nucleotídeoscontendo as proteinasassociadas Anticorposespecificos contra um Fator de Transcriçãopode ser utilizadoparaimunoprecipitar o fragmento de DNA associado a ele O croos-link é revertidoliberandoosfragmentos de danaparasequenciamento Mardis, E.R. Nat. Methods4, 613-614 (2007)

  11. OS FRAGMENTOS DE DNA IMUNOPRECIPTADOS PODEM SER PURIFICADOS E AMPLIFICADOS POR PCR PolII Primers DHFR3’ UTR Primers AdIção de NaHCO3 liberaos anti-corposligadosaoFator de Transcrição Adição de 0.2 M NaCla 67º por3 horasreverte o cross-link proteína/DNA. Os fragmentos de DNA ficamlivresnasolução É adicionadoRNaseA paradigerir RNAs presentesnaamostra Os fragmentos de DNA sãopurificados e amplificadospor PCR)

  12. OS FRAGMENTOS DE DNA SÃO SEQUENCIADOS USANDO-SE SEQUENCIADOR DE 2a GERAÇÃO E OS FRAGMENTOS SÃO MAPEADOS NO CROMOSSOMO

  13. A AÇÃO DE UM FATOR DE TRANSCRIÇÃO OU OUTRA PROTEINA REGULADORA PODE SER MAPEADA EM DIFERENTES TECIDOS ENHANCER-ASSOCIATED PROTEIN P300 Tissue dissection boundaries, overview of the ChIP-seq approach and summary of p300 results. Tissue dissection boundaries are indicated in a representative unstained E11.5 mouse embryo. For each sample, tissue was pooled from more than 150 embryos and ChIP-seq was performed with a p300-antibody. Reads obtained for each of the three tissues that unambiguously aligned to the referencemouse genome were used to define peaks (FDR,0.01). Amore comprehensive overview of sequencing andmapping results is provided in Supplementary Table 1. fb, forebrain; li, limb; mb, midbrain.

  14. FATORES DE TRANSCRIÇÃO SUPERCONSERVADOS PODEM SER ESTUDADOS EM DIFERENTES ESPECIES CEBPA binding in vivo in livers isolated from five vertebrate species cross-mapped to the human PCK1 gene locus. A rare ultraconserved binding event is shown surrounded by speciesspecific and partially shared binding events. On the left is the evolutionary tree of the five study species (Hsap, Homo sapiens; Mmus, Musmusculus; Cfam, Canusfamiliaris; Mdom, Monodelphisdomesticus; Ggal, Gallus gallus), with their approximate evolutionary distance in millions of years ago (MYA). The bottom track shows evolutionary conservation measured across 44 vertebrate species, and darker shading represents slower evolution.

  15. SNPs EM SÍTIOS DE LIGAÇÃO DE FATORES DE TRANSCRIÇÃO PODEM EXPLICAR A VARIAÇÃO FENOTÍPICA ENTRE INDIVÍDUOS DETERMINADA PELO NÍVEL DE TRANSCRIÇÃO DOS GENES REGULADOS POR ELE Effect of SNPs on NFkB and PolII binding. (A) Signal tracks of a NFkB motif and a TATA box demonstrate effects of B-SNPs on TF binding, with correlations in the expected direction (that is, with correct trend). (B) Fold enrichments for cumulative SNP differences affecting BRs and for single SNPs affecting motifs, in pairwise comparisons between individuals relative to the overall frequency of binding differences for NFkB (7.5%) and PolII (25%). (C) B-SNPs affecting motifs frequently lead to binding differences with correct trend. *P < 0.001, based on randomization tests involving 10,000 permutations, that is, permutation tests. (D) BRs adjacent to differentially bound BRs are enriched for binding variation.

  16. TURMA BG-581 2011 DIVIRTAM-SE COM SUAS DESCOBERTAS SOBRE A EXPRESSÃO DIFERENCIAL DOS GENES BOA SORTE