Transcriptional Circuitry of the Yeast Cell Cycle

Yale University, New Haven CT

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Abstract

DESCRIPTION (Applicant?s Abstract) Mitotic entry and progression requires the coordinated expression of numerous genes, many of which are conserved between yeast and higher eukaryotes. While individual transcriptional regulators of the cell cycle have been characterized in yeast, the overall transcriptional circuitry remains to be elucidated. Transcription factor targets can be identified comprehensively, in vivo and on a genome-wide scale through recently developed methodology that combines chromatin immunoprecipitation (ChIP) and DNA microarrays. In order to identify all gene targets of transcription factors that are known or hypothesized to confer cell cycle regulation in yeast, I will: I) Immunoprecipitate transcription factor-bound chromatin and prepare probes from the associated DNA. These probes will be hybridized to a microarray of intergenic regions for the entire yeast genome. 2) Verify transcription factor targets by compiling differential expression profiles for mRNA prepared from wild type and transcription factor mutant strains. 3) Identify consensus sites in promoter regions to find elements involved in cell cycle regulation. Through iterative applications of ChIP experiments and RNA expression studies in wild type and deletion strains, I plan to assemble hierarchies of activators and repressors that comprise the transcriptional circuitry regulating cell division. These techniques are expected to be transferable the study of transcription factors in higher eukaryotes.