Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specific transactivators.
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.
Hearnes
Jamie M
JM
Department of Biochemistry, Center for Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Nashville, TN 37232-6838, USA.
Mays
Deborah J
DJ
Schavolt
Kristy L
KL
Tang
Luojia
L
Jiang
Xin
X
Pietenpol
Jennifer A
JA
D001665:Binding Sites; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D005260:Female; D005557:Formaldehyde / Q000494:pharmacology; D015723:Gene Library; D005821:Genetic Techniques; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D019076:Transgenes; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D002843:Chromatin; D003432:Cross-Linking Reagents; D006133:Growth Substances; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D005557:Formaldehyde; D012313:RNA; D004247:DNA