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      En masse nascent transcription analysis to elucidate regulatory transcription factors

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          Abstract

          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.

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          Analysis of microarray data using Z score transformation.

          Chris Cheadle, Marquis Vawter, William Freed … (2003)
          High-throughput cDNA microarray technology allows for the simultaneous analysis of gene expression levels for thousands of genes and as such, rapid, relatively simple methods are needed to store, analyze, and cross-compare basic microarray data. The application of a classical method of data normalization, Z score transformation, provides a way of standardizing data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridization intensities. Data normalized by Z score transformation can be used directly in the calculation of significant changes in gene expression between different samples and conditions. We used Z scores to compare several different methods for predicting significant changes in gene expression including fold changes, Z ratios, Z and t statistical tests. We conclude that the Z score transformation normalization method accompanied by either Z ratios or Z tests for significance estimates offers a useful method for the basic analysis of microarray data. The results provided by these methods can be as rigorous and are no more arbitrary than other test methods, and, in addition, they have the advantage that they can be easily adapted to standard spreadsheet programs.
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            Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions.

            Sean McCorkle, Soren Impey, Benjamin Dwyer … (2005)
            The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified approximately 41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.
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              Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes.

              O Stevaux, N Dyson, Helen White-Cooper … (2004)
              The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. We have purified E2F/RBF repressor complexes from Drosophila embryo extracts and characterized their roles in E2F regulation. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (publisher-id): Nucleic Acids Research
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date Collection: 2006
                Publication date (Print): 2006
                Publication date (Electronic): 15 March 2006
                Volume: 34
                Issue: 5
                Pages: 1492-1500
                Affiliations
                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
                Author notes
                *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@ 123456nih.gov

                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@ 123456mail.nih.gov

                Article
                DOI: 10.1093/nar/gkj510
                PMC ID: 1408309
                PubMed ID: 16540593
                SO-VID: 49ad2793-0215-4bdf-8b9b-6c0f35038bf2
                Copyright © © The Author 2006. Published by Oxford University Press. All rights reserved
                License:

                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

                History
                Date received : 22 December 2005
                Date revision received : 30 January 2006
                Date accepted : 30 January 2006
                Categories
                Subject: Article

                ScienceOpen disciplines: Genetics
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                ScienceOpen disciplines: Genetics

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