Chromatin immunoprecipitation (ChIP) methodology and readouts.
The identification of direct nuclear hormone receptor gene targets provides clues to their contribution to both development and cancer progression. Until recently, the identification of such direct target genes has relied on a combination of expression analysis and in silico searches for consensus binding motifs in gene promoters. Consensus binding motifs for transcription factors are often defined using in vitro DNA binding strategies. Such in vitro strategies fail to account for the many factors that contribute significantly to target selection by transcription factors in cells beyond the recognition of these short consensus DNA sequences. These factors include DNA methylation, chromatin structure, posttranslational modifications of transcription factors, and the cooperative recruitment of transcription factor complexes. Chromatin immunoprecipitation (ChIP) provides a means of isolating transcription factor complexes in the context of endogenous chromatin, allowing the identification of direct transcription factor targets. ChIP can be combined with site-specific PCR for candidate binding sites or alternatively with cloning, genomic microarrays or more recently direct high throughput sequencing to identify novel genomic targets. The application of ChIP-based approaches has redefined consensus binding motifs for transcription factors and provided important insights into transcription factor biology.
Massie
Charles E
CE
Uro-Oncology Research Group, Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK.
Mills
Ian G
IG
D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation / Q000295:instrumentation / Q000379:methods*; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods; D006801:Humans; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods