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Nucleic Acids Research 2006 34(5):1492-1500; doi:10.1093/nar/gkj510
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Published online 15 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

En masse nascent transcription analysis to elucidate regulatory transcription factors

Jinshui Fan, Ming Zhan1, Jikui Shen2, Jennifer L. Martindale, Xiaoling Yang, Tomoko Kawai and Myriam Gorospe*

Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health Baltimore, MD 21224, USA 1Research Resources Branch, National Institute on Aging-Intramural Research Program, National Institutes of Health Baltimore, MD 21224, USA 2Department of Ophthalmology, Johns Hopkins University School of Medicine Baltimore, MD 21287, USA

*To whom correspondence should be addressed at Box 12, LCMB, NIA-IRP, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Tel: +1 410 558 8443; Fax: +1 410 558 8386; Email: myriam-gorospe{at}nih.gov

Received December 22, 2005. Revised January 30, 2006. Accepted January 30, 2006.

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.

Correspondence may also be addressed to Ming Zhan, RRB, NIA-IRP, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Tel: +1 410 558 8373; Fax: +1 410 558 8674; Email: zhanmi{at}mail.nih.gov


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