Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific Transactivators

Abstract

In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

“…The overlapping sites tended to be the strongest binding sites in both studies (28% of PET3 clusters and 52% of PET5 clusters overlapped with high-confidence ChIP-seq peaks, Table S7A). Similarly, ChIP-seq peaks with height of 31-50 were more likely to overlap with PET3 + clusters than peaks with height of [16][17][18][19][20] (Table S7B). Greater overlap between the higher ranked sites is expected in genome-wide studies.…”

“…3,17 Recent advances in sequencing technologies combined with chromatin immunoprecipitation (ChIP) have lead to the identification of thousands of p53 binding sites, providing unmatched opportunities for analysis and comparison of the global genomic p53 binding pattern under different experimental conditions. Notably, all de novo p53 binding studies published to date [18][19][20][21][22][23][24][25] have used cancer-derived cell lines. Binding of transcription factors (TF) to DNA is known to be context-dependent, governed in vivo by chromatin architecture and epigenetic modifications, 26 and these are subjected to major changes during tumor development.…”

p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm

“…qRT-PCR HMEC transduction, mRNA preparation and subsequent qRT-PCR studies were carried out as described previously (Hearnes et al, 2005). Primers are listed in Supplementary Table 6.…”

“…HMECs were cultured in the presence or absence of 350 nM ADR for 6 h and chromatin was prepared as described previously (Hearnes et al, 2005). The ChIP and qRT-PCR primers are described in Supplementary Table 6.…”

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4α (HNF4α) gene

“…This indicates that there may be other more divergent p53 binding sites in the promoter that mediate repression. Others have noted that the p53 response elements involved in transcriptional repression do not fit the consensus as well as those involved in activation [3,37].…”