The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate the following basic issues concerning molecular mechanisms of transcriptional regulation and chromatin structure in yeast, by combining molecular genetic, biochemical, functional genomic, and evolutionary approaches. First, we will investigate the molecular mechanisms by which the in vivo pattern of nucleosomes is established. We will a) identify heretofore unknown factors involved in nucleosome depletion at terminators, B) define DNA sequence requirements for nucleosome formation by selecting high affinity sites from randomized 147 bp fragments, C) functionally evaluate and mechanistically elucidate a transcription-based mechanism for statistical positioning of nucleosomes in coding regions, and D) define determinants of nucleosome occupancy and positioning by comparing the nucleosome patterns of genomic DNA from diverse yeast species in S. cerevisiae vs. nucleosome patterns in the endogenous organism. Second, we will use the anchor-away method for rapidly removing proteins from the nucleus to investigate fundamental aspects of Pol II transcriptional initiation and elongation including A) the nature and stability of the preinitiation complex, B) the role of CF1 (a complex involved in 3'end formation and termination) in initiation and elongation, C) the role of FACT and Spt6 in Pol II transcription and possible nucleosome eviction, and D) detailed examination of elongation factors for their functions in vivo. In addition, we will examine E) the unexpected role of histone H1 in transcription, F) the issue of whether Pol II elongation factors travel in a large complex throughout the coding regions, G) and the effect of temperature, growth conditions, and specific transcriptional regulatory proteins on the general relationship between Pol II transcription and RNA half-life. Third, we will use a novel conceptual and experimental approach to distinguish biological function from biological noise that is based on a comparison of physiological responses, RNA and transcription factor binding profiles, and effects of mutations in yeast species of varying evolutionary distance. We will explicitly measure biological noise by making functional measurements of evolutionary irrelevant or random-sequence DNA in yeast. Overall, the proposal will answer fundamental questions and address previously unknown aspects about the highly interlinked processes of Pol II transcription and nucleosome occupancy and positioning, as well as pioneer a new approach about distinguishing biological function from biological noise.

Public Health Relevance

Regulation of gene expression is a critical aspect of many biological phenomena (e.g. cell growth, development of multicellular organisms, the response to environmental conditions, and evolution), and alteration of normal gene regulation can lead to human disease. This proposal investigates fundamental molecular mechanisms of gene regulation at several levels, and it also uses a novel functional evolutionary approach to understand broad issues related to biological function. The results will continue to shed new light on fundamental issues in gene regulation and will have a significant impact on our understanding of human biology and disease.

National Institute of Health (NIH)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Sledjeski, Darren D
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Harvard Medical School
Schools of Medicine
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Geisberg, Joseph V; Moqtaderi, Zarmik; Fan, Xiaochun et al. (2014) Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast. Cell 156:812-24
Wong, Koon Ho; Jin, Yi; Struhl, Kevin (2014) TFIIH phosphorylation of the Pol II CTD stimulates mediator dissociation from the preinitiation complex and promoter escape. Mol Cell 54:601-12
Moqtaderi, Zarmik; Geisberg, Joseph V; Struhl, Kevin (2014) Secondary structures involving the poly(A) tail and other 3' sequences are major determinants of mRNA isoform stability in yeast. Microb Cell 1:137-139
Moqtaderi, Zarmik; Geisberg, Joseph V; Jin, Yi et al. (2013) Species-specific factors mediate extensive heterogeneity of mRNA 3' ends in yeasts. Proc Natl Acad Sci U S A 110:11073-8
Fleming, Joseph D; Pavesi, Giulio; Benatti, Paolo et al. (2013) NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res 23:1195-209
Struhl, Kevin; Segal, Eran (2013) Determinants of nucleosome positioning. Nat Struct Mol Biol 20:267-73
Esberg, Anders; Moqtaderi, Zarmik; Fan, Xiaochun et al. (2011) Iwr1 protein is important for preinitiation complex formation by all three nuclear RNA polymerases in Saccharomyces cerevisiae. PLoS One 6:e20829
Fan, Xiaochun; Geisberg, Joseph V; Wong, Koon Ho et al. (2011) Conditional depletion of nuclear proteins by the Anchor Away system. Curr Protoc Mol Biol Chapter 13:Unit13.10B
Miotto, Benoit; Struhl, Kevin (2011) JNK1 phosphorylation of Cdt1 inhibits recruitment of HBO1 histone acetylase and blocks replication licensing in response to stress. Mol Cell 44:62-71
Wong, Koon Ho; Struhl, Kevin (2011) The Cyc8-Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein. Genes Dev 25:2525-39

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