RESUMO
The ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.
Assuntos
Mapeamento Cromossômico , Genoma Humano , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Sítios de Ligação/genética , Imunoprecipitação da Cromatina/métodos , DNA/análise , Células HCT116 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The protooncogene MYC encodes the c-Myc transcription factor that regulates cell growth, cell proliferation, cell cycle, and apoptosis. Although deregulation of MYC contributes to tumorigenesis, it is still unclear what direct Myc-induced transcriptomes promote cell transformation. Here we provide a snapshot of genome-wide, unbiased characterization of direct Myc binding targets in a model of human B lymphoid tumor using ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET). Myc potentially occupies > 4,000 genomic loci with the majority near proximal promoter regions associated frequently with CpG islands. Using gene expression profiles with ChIP-PET, we identified 668 direct Myc-regulated gene targets, including 48 transcription factors, indicating that Myc is a central transcriptional hub in growth and proliferation control. This first global genomic view of Myc binding sites yields insights of transcriptional circuitries and cis regulatory modules involving Myc and provides a substantial framework for our understanding of mechanisms of Myc-induced tumorigenesis.