Interactions between transcription factors (TFs) and their DNA binding sites are an integral part of regulatory networks within cells. These interactions control critical steps in progression through normal cellular processes and in responses to various environmental stresses. However, the DNA binding site specificities and regulatory functions of many known and most predicted TFs in S. cerevisiae are unknown. The Bulyk Lab has recently developed an improved in vitro protein binding microarray (PBM) technology to characterize TFs' sequence specificities in a high-throughput manner. The PBM technology allows us to determine the binding site specificity of known or predicted transcription factors in a single day, starting from the purified TF. Preliminary comparisons of binding site specificities determined from the PBM approach correspond well with binding site specificities determined by in vivo genome-wide location analysis. We propose to characterize the DNA binding site specificities of all known and predicted TFs whose binding specificities are as yet unknown. To achieve this goal, the Bulyk Lab has teamed up with Dr. Joshua Labaer's Lab (Harvard Medical School and the Harvard Institute of Proteomics), whose expertise with highthroughput cloning, expression, and purification will enable us to examine a collection of ~300 candidate yeast TFs, and with Dr. Richard Young (MIT / Whitehead Institute) and the Whitehead Institute's Microarray Center, who will be providing whole-genome yeast intergenic microarrays for use in PBM experiments. The TFs' binding sites will enable us to predict the TFs' functional roles by examining the functions of the candidate target genes. These candidate functional roles will be examined experimentally by assessing the phenotypes of the corresponding TF deletion strains under conditions in which the particular TFs are hypothesized to regulate the candidate target genes. Comparison of the in vitro PBM data and the in vivo genome-wide location analysis data may provide data as to why certain sites are or are not used in vivo. These studies will also serve as a model system for applying the PBM technology on a genome- and proteome-wide scale. In the future this kind of scaled-up PBM approach could be used to examine a large collection of candidate combinatorial TF interactions in DNA binding. This PBM approach could also be used to study TFs from other genomes. We will make our data publicly available in a web-accessible database.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
3R01HG003420-03S1
Application #
7654781
Study Section
Genome Study Section (GNM)
Program Officer
Feingold, Elise A
Project Start
2004-09-24
Project End
2009-03-31
Budget Start
2006-09-01
Budget End
2009-03-31
Support Year
3
Fiscal Year
2008
Total Cost
$265,151
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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Gordan, Raluca; Hartemink, Alexander J; Bulyk, Martha L (2009) Distinguishing direct versus indirect transcription factor-DNA interactions. Genome Res 19:2090-100
Zhu, Cong; Byers, Kelsey J R P; McCord, Rachel Patton et al. (2009) High-resolution DNA-binding specificity analysis of yeast transcription factors. Genome Res 19:556-66
McCord, Rachel Patton; Berger, Michael F; Philippakis, Anthony A et al. (2007) Inferring condition-specific transcription factor function from DNA binding and gene expression data. Mol Syst Biol 3:100
Hu, Yanhui; Rolfs, Andreas; Bhullar, Bhupinder et al. (2007) Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae. Genome Res 17:536-43
Bulyk, Martha L (2006) Analysis of sequence specificities of DNA-binding proteins with protein binding microarrays. Methods Enzymol 410:279-99
Berger, Michael F; Philippakis, Anthony A; Qureshi, Aaron M et al. (2006) Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol 24:1429-35
Berger, Michael F; Bulyk, Martha L (2006) Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins. Methods Mol Biol 338:245-60
Bulyk, Martha L (2006) DNA microarray technologies for measuring protein-DNA interactions. Curr Opin Biotechnol 17:422-30
Lieb, J D; Beck, S; Bulyk, M L et al. (2006) Applying whole-genome studies of epigenetic regulation to study human disease. Cytogenet Genome Res 114:1-15

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