Nucleic Acids Research Advance Access originally published online on December 17, 2007
Nucleic Acids Research 2008 36(1):e8; doi:10.1093/nar/gkm1117
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Nucleic Acids Research, 2008, Vol. 36, No. 1 e8
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Genome wide screens in yeast to identify potential binding sites and target genes of DNA-binding proteins
1Division of Hematology/Oncology, Department of Medicine, University of California, Irvine, CA, 2Genome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, MD, 3Howard Hughes Medical Institute, Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA, 4Department of Genetics, Washington University School of Medicine, Saint Louis, MO, 5Departments of Computer Science and Biomedical Engineering, 6Institute for Genomics and Bioinformatics and 7Departments of Biological Chemistry and Pathology & Laboratory Medicine, University of California, Irvine, CA, USA
*To whom correspondence should be addressed: Tel: +301 594 8224; Fax: +301 496 0474; Email: burgess{at}mail.nih.gov
Received October 24, 2007. Revised November 28, 2007. Accepted November 29, 2007.
Knowledge of all binding sites for transcriptional activators and repressors is essential for computationally aided identification of transcriptional networks. The techniques developed for defining the binding sites of transcription factors tend to be cumbersome and not adaptable to high throughput. We refined a versatile yeast strategy to rapidly and efficiently identify genomic targets of DNA-binding proteins. Yeast expressing a transcription factor is mated to yeast containing a library of genomic fragments cloned upstream of the reporter gene URA3. DNA fragments with target-binding sites are identified by growth of yeast clones in media lacking uracil. The experimental approach was validated with the tumor suppressor protein p53 and the forkhead protein FoxI1 using genomic libraries for zebrafish and mouse generated by shotgun cloning of short genomic fragments. Computational analysis of the genomic fragments recapitulated the published consensus-binding site for each protein. Identified fragments were mapped to identify the genomic context of each binding site. Our yeast screening strategy, combined with bioinformatics approaches, will allow both detailed and high-throughput characterization of transcription factors, scalable to the analysis of all putative DNA-binding proteins.
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.
Present addresses: Rainer K. Brachmann, Genentech, Inc.1 DNA Way, MS443B South San Francisco, CA, USA Jizhou Yan, Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA
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