En masse nascent transcription analysis to elucidate regulatory transcription factors.
Despite exhaustively informing about steady-state mRNA abundance, DNA microarrays have been used with limited success to identify regulatory transcription factors (TFs). The main limitation of this approach is that altered mRNA stability also strongly governs the patterns of expressed genes. Here, we used nuclear run-on assays and microarrays to systematically interrogate changes in nascent transcription in cells treated with the topoisomerase inhibitor camptothecin (CPT). Analysis of the promoters of coordinately transcribed genes after CPT treatment suggested the involvement of TFs c-Myb and Rfx1. The predicted CPT-dependent associations were subsequently confirmed by chromatin immunoprecipitation assays. Importantly, after RNAi-mediated knockdown of each TF, the CPT-elicited induction of c-Myb- and/or Rfx1-regulated mRNAs was diminished and the overall cellular response was impaired. The strategies described here permit the successful identification of the TFs responsible for implementing adaptive gene expression programs in response to cellular stimulation.
Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA.
Jinshui
J
Fan
Ming
M
Zhan
Jikui
J
Shen
Jennifer L
JL
Martindale
Xiaoling
X
Yang
Tomoko
T
Kawai
Myriam
M
Gorospe
D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D020598:Proto-Oncogene Proteins c-myb / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D020598:Proto-Oncogene Proteins c-myb; C000605537:RFX1 protein, human; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D014157:Transcription Factors; D002166:Camptothecin