Regulation of gene expression is essential for the proper differentiation and maintenance of distinct cell types in eukaryotes. A detailed understanding of transcriptional regulation is important for medicine because specific diseases, including cancer, can result from abnormalities in gene expression. The yeast Saccharomyces cerevisiae has proven to be a model eukaryotic organism for many types of studies, including gene regulation. Importantly, the transcription regulatory machinery is conserved between yeast and vertebrates, and insights gained from studies in yeast are generally universal. Studying the transcriptional regulation of the yeast HO gene has been very productive in terms of identifying important factors and novel regulatory paradigms that are conserved in metazoans. Using chromatin immunoprecipitation (ChIP) methods with synchronized cells, we have made several novel observations about regulatory events at the HO promoter. 1. Nucleosome eviction occurs in waves both during the cell cycle and along the length of the promoter. 2. Accompanying these changes in promoter structure, three coactivator complexes, Swi/Snf, SAGA, and Mediator, all bind first to the far upstream promoter region and subsequently to a promoter proximal region. 3. These changes in chromatin structure and coactivator binding are dependent on other chromatin factors, including the FACT chromatin reorganizing complex and the Asf1 histone chaperone, and specific factors are required for changes in chromatin structure at distinct promoter regions. Finally, these changes occur in the absence of RNA pol II.
The specific aims of this proposal are to understand the molecular mechanisms underlying these changes in promoter structure during the cell cycle. This regulatory paradigm may be seen at other genes with complex regulation. In the upcoming grant cycle experiments will be performed to decipher the molecular mechanisms underlying these chromatin transitions that occur in waves along the promoter during the cell cycle. The HO promoter has two distinct regions, each with multiple binding sites for distinct sequence-specific DNA-binding factors. Our data suggest may be communication between the two promoter regions, and this idea will be tested directly. We will determine how the FACT chromatin reorganizing complex is recruited to the HO promoter, and whether other chromatin regulators are involved in the chromatin transitions. The program of chromatin transitions is initiated by binding of the Swi5 DNA-binding factor. Swi5 recruits coactivators to the upstream region and then Swi5 is degraded. Remarkably, there is no Swi5 protein bound to the HO promoter at the time the gene is transcribed. Thus cells must have a """"""""memory"""""""" that Swi5 was present, and this memory persists for a remarkably long time. We will determine whether persistent changes in chromatin structure represent this memory mark, and how such a chromatin mark is generated and maintained.

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Project Narrative: Regulation of gene expression is essential for the proper differentiation and maintenance of distinct cell types in eukaryotes, and abnormalities in gene expression can cause specific diseases including cancer. The yeast Saccharomyces cerevisiae with its powerful genetic tools has proven to be the model eukaryotic organism for studies of gene regulation, and since the transcription regulatory machinery is conserved between yeast and vertebrates the insights gained from studies in yeast are generally universal. Here we study the molecular mechanisms underlying the complex changes that occur in the chromatin structure at a promoter preceding transcriptional activation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Carter, Anthony D
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Yarrington, Robert M; Goodrum, Jenna M; Stillman, David J (2016) Nucleosomes Are Essential for Proper Regulation of a Multigated Promoter in Saccharomyces cerevisiae. Genetics 202:551-63
Yu, Yaxin; Yarrington, Robert M; Chuong, Edward B et al. (2016) Disruption of promoter memory by synthesis of a long noncoding RNA. Proc Natl Acad Sci U S A 113:9575-80
Yarrington, Robert M; Rudd, Jared S; Stillman, David J (2015) Spatiotemporal cascade of transcription factor binding required for promoter activation. Mol Cell Biol 35:688-98
Zapata, Jessica; Dephoure, Noah; Macdonough, Tracy et al. (2014) PP2ARts1 is a master regulator of pathways that control cell size. J Cell Biol 204:359-76
Parnell, Emily J; Yu, Yaxin; Lucena, Rafael et al. (2014) The Rts1 regulatory subunit of PP2A phosphatase controls expression of the HO endonuclease via localization of the Ace2 transcription factor. J Biol Chem 289:35431-7
Voth, Warren P; Takahata, Shinya; Nishikawa, Joy L et al. (2014) A role for FACT in repopulation of nucleosomes at inducible genes. PLoS One 9:e84092
Stillman, David J (2013) Dancing the cell cycle two-step: regulation of yeast G1-cell-cycle genes by chromatin structure. Trends Biochem Sci 38:467-75
Zhang, Qian; Yoon, Youngdae; Yu, Yaxin et al. (2013) Stochastic expression and epigenetic memory at the yeast HO promoter. Proc Natl Acad Sci U S A 110:14012-7
Tantin, Dean; Voth, Warren P; Shakya, Arvind (2013) Efficient chromatin immunoprecipitation using limiting amounts of biomass. J Vis Exp :e50064
Stillman, David J (2010) Nhp6: a small but powerful effector of chromatin structure in Saccharomyces cerevisiae. Biochim Biophys Acta 1799:175-80

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