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 histonemodifying acetyltransferase 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 histone acetylation (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 complexcontaining 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 formaldeido faz o “cross-link” das moleculas de proteinas ao DNA “congelando por exemplo os Fatores de Transcrição no seu sítio de ligação ao DNACross-linking pode ser feito em:Celulas em suspensãoCultura de células em placaTecidosCortes histológicos
10 O ISOLAMENTO DO DNA GENOMICO CARREGA AS ROTEINAS ASSOCIADAS Após sonicação são produzidos fragmentos de DNA de ~ 500 nucleotídeos contendo as proteinas associadasAnticorpos especificos contra um Fator de Transcrição pode ser utilizado para imunoprecipitar o fragmento de DNA associado a eleO croos-link é revertido liberando os fragmentos de dana para sequenciamentoMardis, E.R. Nat. Methods 4, (2007)
11 OS FRAGMENTOS DE DNA IMUNOPRECIPTADOS PODEM SER PURIFICADOS E AMPLIFICADOS POR PCR AdIção de NaHCO3 libera os anti-corpos ligados ao Fator de TranscriçãoAdição de 0.2 M NaCl a 67º por 3 horas reverte o cross-link proteína/DNA. Os fragmentos de DNA ficam livres na soluçãoÉ adicionado RNase A para digerir RNAs presentes na amostraOs fragmentos de DNA são purificados e amplificados por PCR)PolII PrimersDHFR3’ UTR Primers
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 TECIDOSENHANCER-ASSOCIATED PROTEIN P300Tissue 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, Mus musculus; Cfam, Canus familiaris; Mdom, Monodelphis domesticus; Ggal, Gallus gallus), with their approximate evolutionary distance in millions of years ago (MYA). Thebottom track shows evolutionary conservation measured across 44 vertebrate species, and darker shading representsslower 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 ELEEffect of SNPs on NFkB and PolII binding. (A) Signal tracks of a NFkB motif and a TATA boxdemonstrate 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 differencesfor 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 BOA SORTE TURMA BG-581 2011 DIVIRTAM-SE COM SUAS DESCOBERTAS SOBRE A EXPRESSÃO DIFERENCIAL DOS GENESBOA SORTE
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