The long term objectives of this application are to learn more about how chromatin structure controls transcription initiation in eukaryotic cells. Histones and certain classes of transcription factors that control chromatin structures have been demonstrated to play essential roles in normal transcription in all eukaryotes, ranging from yeast to humans. Mutations that impair the function of these factors have been associated with human diseases. The studies in this proposal, comprised of both genetic and biochemical approaches, focus on four sets of factors and how they function in vivo in the yeast, Saccharomyces cerevisiae. First, studies of the Snf/Swi complex will be conducted. Snf/Swi, a complex of twelve proteins, is believed to control transcription by remodeling nucleosomes, the fundamental unit of chromatin. The proposed studies will address the precise role of Snf/Swi at a promoter, the requirement for Snf/Swi for transcription of all S. cerevisiae genes, and the classes of mutants that impair their interaction with Snf/Swi. The mutants will be characterized genetically. The mutant histones will be purified and used to assemble mutant nucleosomes to be studied in vitro with respect to interaction with Snf/Swi and other factors. Third, studies of the Spt6 protein will be pursued. Spt6 is an essential protein, known to interact with histones. Spt6 and any associated proteins will be pursued. Spt6 is an essential protein, known to interact with histones. Spt6 and any associated proteins will be purified from S. cerevisiae cells. The associated proteins will be identified biochemically and studied genetically. To identify the role of particular motifs in Spt6, deletion mutations will be studied in vivo and in vitro. Finally, studies will be pursued of a putative histone acetyltransferase (HAT), Spt10. Spt10 is strongly required for the transcription of particular histone genes and its sequence suggests that it is a HAT. Site-directed mutations in the HAT consensus region of the SPT10 gene will be tested for function in vivo. HAT assays will be

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032967-18
Application #
6329646
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Carter, Anthony D
Project Start
1983-12-01
Project End
2003-11-30
Budget Start
2000-12-01
Budget End
2001-11-30
Support Year
18
Fiscal Year
2001
Total Cost
$371,740
Indirect Cost
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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Winston, Fred; Koshland, Douglas (2016) Back to the Future: Mutant Hunts Are Still the Way To Go. Genetics 203:1007-10
DeGennaro, Christine M; Alver, Burak H; Marguerat, Samuel et al. (2013) Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast. Mol Cell Biol 33:4779-92
Chang, Jennifer S; Winston, Fred (2013) Cell-cycle perturbations suppress the slow-growth defect of spt10? mutants in Saccharomyces cerevisiae. G3 (Bethesda) 3:573-83
Rando, Oliver J; Winston, Fred (2012) Chromatin and transcription in yeast. Genetics 190:351-87
Kiely, Christine M; Marguerat, Samuel; Garcia, Jennifer F et al. (2011) Spt6 is required for heterochromatic silencing in the fission yeast Schizosaccharomyces pombe. Mol Cell Biol 31:4193-204
Ivanovska, Iva; Jacques, Pierre-Étienne; Rando, Oliver J et al. (2011) Control of chromatin structure by spt6: different consequences in coding and regulatory regions. Mol Cell Biol 31:531-41
Chang, Jennifer S; Winston, Fred (2011) Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae. Eukaryot Cell 10:118-29
Libuda, Diana E; Winston, Fred (2010) Alterations in DNA replication and histone levels promote histone gene amplification in Saccharomyces cerevisiae. Genetics 184:985-97

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