. The goal of this project is to define the interactions between RNA polymerase II, the basal transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using a combination of approaches in the yeast Saccharomyces cerevisiae model system, several fundamental aspects of gene expression will be studied. In the first specific aim, initiation and elongation complexes formed under various conditions will be purified on immobilized chromatin templates and then analyzed by quantitative mass spectrometry. It is possible some new factors associated with transcription complexes will be identified, but what is of greater interest is the exchange of factors that occur at various stages of transcription. Using various mutant or depleted extracts, factor interdependencies will be revealed. We will ask whether different activators recruit distinct co-activators, and how different promoters and transcribed sequences influence the associated proteins. The second specific aim will probe the function of the Set1/COMPASS complex, which methylates histone H3 at lysine 4 (H3K4). Methylation is co- transcriptional, and H3K4 trimethylation is strongest at the nucleosome nearest the transcription start site, while dimethylation predominates further downstream through the next few nucleosomes. We will explore how Set1/COMPASS is recruited to transcription complexes and how this gradient of methylation is achieved.
This aim will also address how levels of COMPASS are controlled. The third specific aim of this project will study the downstream effects of the co-transcriptional histone methylations at H3K4 and H3K36. We will continue our work on the Set3 histone deacetylase complex, which recognizes H3K4me2, and the Rpd3S histone deacetylase complex that recognizes H3K36me2/3. Preliminary data suggests that the Set3 complex strongly affects the kinetics of transcription induction and repression, and several molecular models to explain these effects will be tested. The role of H3K4me3 in recruiting various chromatin modifying complexes will also be explored. The experiments in these three specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects.

Public Health Relevance

Project Narrative. Improper gene expression causes many diseases, including developmental defects and cancer. The goal of this project is to understand the fundamental processes by which genes are expressed and regulated. This understanding will be essential for designing treatments and drugs to restore normal gene expression in diseased cells or to alter gene expression patterns to create pluripotent stem cells and specific differentiated cell types.

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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Sledjeski, Darren D
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Harvard University
Schools of Medicine
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Joo, Yoo Jin; Ficarro, Scott B; Soares, Luis M et al. (2017) Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation. Genes Dev 31:2162-2174
Woo, Hyeonju; Dam Ha, So; Lee, Sung Bae et al. (2017) Modulation of gene expression dynamics by co-transcriptional histone methylations. Exp Mol Med 49:e326
Soares, Luis M; He, P Cody; Chun, Yujin et al. (2017) Determinants of Histone H3K4 Methylation Patterns. Mol Cell 68:773-785.e6
Kim, Ji Hyun; Lee, Bo Bae; Oh, Young Mi et al. (2016) Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway. Nat Commun 7:13534
Hahn, Steven; Buratowski, Stephen (2016) Structural biology: Snapshots of transcription initiation. Nature 533:331-2
Soares, Luis M; Radman-Livaja, Marta; Lin, Sherry G et al. (2014) Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns. Cell Rep 6:961-972
Marquardt, Sebastian; Escalante-Chong, Renan; Pho, Nam et al. (2014) A chromatin-based mechanism for limiting divergent noncoding transcription. Cell 157:1712-23
Suh, Hyunsuk; Hazelbaker, Dane Z; Soares, Luis M et al. (2013) The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol Cell 51:850-8
Soares, Luis M; Buratowski, Stephen (2013) Histone Crosstalk: H2Bub and H3K4 Methylation. Mol Cell 49:1019-20
van Werven, Folkert J; Neuert, Gregor; Hendrick, Natalie et al. (2012) Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in budding yeast. Cell 150:1170-81

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