The long-term objectives of this proposal are to elucidate mechanisms for controlling transcription initiation and elongation in eukaryotes. The four specific aims are focused on distinct sets of transcription factors, many of which are highly conserved from yeast to human. These studies will be performed using the yeast Saccharomyces cerevisiae, and multiple experimental approaches will be used. The factors to be studied are known to be either essential or critical for normal transcription in vivo. First, a nucleosome remodeling complex, Swi/Snf, will be studied with respect to its role in transcriptional repression. Experiments will be done to test the hypothesis that Swi/Snf repression occurs after transcription initiation by RNA polymerase II (RNAP II), including chromatin immunoprecipitation (CHIP), potassium permanganate footprinting, and transcription run-on experiments. Other factors required for repression with Swi/Snf will be identified by mutant screens and selections. Second, the elongation factor Spt6 will be studied with respect to (1) its positive role in elongation and (2) a role in repression of transcription initiation at cryptic promoters within open reading frames. These experiments will use CHIP, chromatin structure analysis, and mutant selections. Third, a previously unstudied transcription elongation factor, named Elf1, will be analyzed by ChIP experiments to understand its interactions with other elongation factors. In addition, an in vitro assay will be used to test for Elf1' s direct role in elongation. The role of Elf1 in vivo will be addressed by whole-genome expression analysis using DNA microarrays. Fourth, Spt10, a putative histone acetyltransferase, will be studied with respect to its roles in transcription of histone genes and nonhistone genes. Its role at histone gene promoters will be studied by ChIP experiments. These assays will include analysis of the Spt10- interacting protein Spt21. Genetic selections will be done to identify other factors that interact with Spt10 and Spt21. Finally, a combination of microarray and ChIP experiments will identify other genes directly controlled by Spt10. Given the strong conservation of transcriptional mechanisms throughout eukaryotes, the knowledge gained from these studies will be directly relevant to understanding transcription in humans. These studies are relevant to human disease, since mutations that impair human nucleosome remodeling complexes, HATs, and other types of factors have been associated with certain diseases including cancer. ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG1-MBC-2 (01))
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Carter, Anthony D
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Harvard University
Schools of Medicine
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