The long-term objectives of the proposed research are to identify proteins that play general and important roles in transcription by RNA polymerase II (pol II) and to elucidate the mechanisms by which these proteins act. This grant focuses on the highly regulated process of transcription elongation. While a number of eukaryotic transcription elongation factors have been identified in recent years, there is little mechanistic understanding of how these proteins facilitate or impede RNA synthesis in the context of chromatin.
Specific Aims 1 and 2 focus on the conserved Paf1 complex, which interacts with RNA pol II and couples chromatin changes to transcription elongation.
Specific Aim1 is to determine the mechanism by which the Paf1 complex directs histone modifications. Genetic suppressor and biochemical screens will be used to identify proteins that interact with a recently identified histone modification domain in the Rtf1 subunit of the Paf1 complex. Experiments will be performed to test whether this domain is sufficient to establish histone H2B ubiquitylation and histone H3 lysine 4 and 79 methylation in the absence of ongoing transcription. The role of the Paf1 complex in regulating two additional histone modifications on active genes, histone H3 lysine 36 tri-methylation and histone acetylation, will be studied. Candidate gene approaches and genetic selections will be performed to reveal the histone acetyltransferase(s) and histone deacetylase(s) that collaborate with the Paf1 complex to repress histone acetylation and spurious transcription initiation within coding regions.
Specific Aim 2 is to investigate three unexplored transcriptional functions of the Paf1 complex. The function of the Paf1 complex in gene repression will be studied using ARG1 as a model gene. The molecular pathway to Paf1 complex- dependent repression and the impact of the Paf1 complex on histone modification and nucleosome positioning will be investigated. Mutations in PAF1 that specifically alleviate ARG1 repression will be identified to expose the molecular basis of repression. Our recent genome tiling array studies revealed broad effects of the Paf1 complex on gene expression. These data will be analyzed further, and two specific transcriptional patterns, which indicate a requirement for the Paf1 complex in overcoming elongation blocks and in repressing cryptic transcription initiation, will be studied in detail.
Specific Aim 3 is to investigate the function of Rkr1, a novel nuclear ubiquitin-protein ligase that exhibits strong functional connections to the Paf1 complex and other proteins required for proper chromatin function. Experiments in this Aim test the hypothesis that Rkr1 regulates the modification and function of a histone variant that has conserved and important roles in transcription. Defects in the activity or expression of the human Paf1 complex and the SET domain proteins with which it interacts are associated with multiple cancers, including pancreatic cancer, hyperparathyroidism- jaw tumor syndrome, and leukemia. Therefore, the results of the proposed studies are expected to yield insights into the causes of several types of cancer that afflict humans.

Public Health Relevance

Human cancers arise when genetic mutations lead to uncontrolled cell growth. These mutations alter the activities of proteins that are critical for the proper regulation of cell division. Mutations that change the activity of the Paf1 complex, the primary focus of this application, and the SET-domain proteins, which interact with the Paf1 complex, lead to the improper expression of genes involved in cell cycle control and are thus associated with multiple types of cancer, including pancreatic cancer, leukemia, and hyperparathyroidism-jaw tumor syndrome.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Sledjeski, Darren D
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University of Pittsburgh
Schools of Arts and Sciences
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Wier, Adam D; Mayekar, Manasi K; Heroux, Annie et al. (2013) Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin. Proc Natl Acad Sci U S A 110:17290-5
Tomson, Brett N; Crisucci, Elia M; Heisler, Lawrence E et al. (2013) Effects of the Paf1 complex and histone modifications on snoRNA 3'-end formation reveal broad and locus-specific regulation. Mol Cell Biol 33:170-82
Tomson, Brett N; Arndt, Karen M (2013) The many roles of the conserved eukaryotic Paf1 complex in regulating transcription, histone modifications, and disease states. Biochim Biophys Acta 1829:116-26
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