The long-term objectives of the proposed research are to identify the proteins and mechanisms that regulate transcription by RNA polymerase II (pol II) particularly within the context of chromatin. The focus of this proposal is on the globally acting, multi-functional Paf1 complex (Paf1C). Paf1C associates with RNA pol II during transcription elongation, coupling critical events to RNA synthesis. These events include histone modification and efficient transcription termination and RNA 3'-end formation. The broad impact of Paf1C on gene expression highlights the significance of the research plan.
Specific Aim 1 is to uncover the mechanistic basis for the requirement of Paf1C in histone H2B K123 mono-ubiquitylation. This modification marks active genes, regulates transcription, and controls other key epigenetic marks on chromatin. Biochemical and genetic studies will be used to identify proteins that interact with a domain of Paf1C that is both necessary and sufficient for H2B K123 ubiquitylation. Enzymatic and genomic experiments will test the impact of Paf1C on H2B K123 ubiquitylation in vitro and in vivo. In addition, the role of an exposed nucleosomal surface in regulating H2B K123 ubiquitylation and downstream methylation marks on H3 will be probed.
Specific Aim 2 is to determine the mechanisms that couple Paf1C to RNA pol II during elongation. Absence of this coupling leads to severe mutant phenotypes, indicative of broad disruptions in transcription. Protein interaction and genetic studies will be used to identify the proteins that tether Paf1C to the RNA pol II elongation machinery and regulate this interaction. Mutants will be exploited to disrupt the Paf1C-RNA pol II interaction, and the consequences of these mutants on histone modification, RNA pol II phosphorylation, and transcript synthesis will be assessed using targeted and genomic strategies. Mutants designed to interfere with Paf1C dissociation from RNA pol II will be used to test the importance of the conserved localization pattern of Paf1C on genes.
Specific Aim 3 is to elucidate the role of Paf1C in regulating transcription termination and synthesis of noncoding RNAs, including snoRNAs and cryptic unstable transcripts. Molecular experiments will determine the mechanisms by which the chromatin-related functions of Paf1C impact termination efficiency at snoRNA genes. Tiling array studies will be used to determine the scope of the involvement of Paf1C, and relative contributions of its activities, in regulating synthesis of snoRNAs and cryptic unstable transcripts. Finally, a comprehensive genetic screen will be performed to systematically interrogate the role of histones in transcription termination. The work will be performed in yeast to exploit the powerful genetic tools available in this system and because extensive conservation exists between the yeast and human Paf1 complexes. Genetic perturbations that deregulate Paf1C are associated with multiple types of human cancers, including those of pancreatic, breast, uterine, and thyroid origin. The results of the proposed studies are therefore expected to have important and broad consequences for understanding the causes of cancer.

Public Health Relevance

Cancer arises when genetic mutations lead to uncontrolled cell growth. These genetic mutations alter the activities of proteins that normally control the division of cells. Mutations that impairthe function of the highly conserved Paf1 complex (Paf1C), the focus of this application, and the SET-domain proteins, with which Paf1C interacts, deregulate the expression of genes involved in cell cycle control and are thus associated with multiple human cancers, including pancreatic cancer, breast cancer, uterine cancer, leukemia, and hyperparathyroidism-jaw tumor syndrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052593-19
Application #
8716766
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
1995-05-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
19
Fiscal Year
2014
Total Cost
$391,975
Indirect Cost
$128,402
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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
Mayekar, Manasi K; Gardner, Richard G; Arndt, Karen M (2013) The recruitment of the Saccharomyces cerevisiae Paf1 complex to active genes requires a domain of Rtf1 that directly interacts with the Spt4-Spt5 complex. Mol Cell Biol 33:3259-73
Amrich, Christopher G; Davis, Christopher P; Rogal, Walter P et al. (2012) Cdc73 subunit of Paf1 complex contains C-terminal Ras-like domain that promotes association of Paf1 complex with chromatin. J Biol Chem 287:10863-75
Crisucci, Elia M; Arndt, Karen M (2012) Paf1 restricts Gcn4 occupancy and antisense transcription at the ARG1 promoter. Mol Cell Biol 32:1150-63
Tomson, Brett N; Davis, Christopher P; Warner, Marcie H et al. (2011) Identification of a role for histone H2B ubiquitylation in noncoding RNA 3'-end formation through mutational analysis of Rtf1 in Saccharomyces cerevisiae. Genetics 188:273-89
Crisucci, Elia M; Arndt, Karen M (2011) The Paf1 complex represses ARG1 transcription in Saccharomyces cerevisiae by promoting histone modifications. Eukaryot Cell 10:712-23
Rubenstein, Eric M; McCartney, Rhonda R; Zhang, Chao et al. (2008) Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase. J Biol Chem 283:222-30
Shirra, Margaret K; McCartney, Rhonda R; Zhang, Chao et al. (2008) A chemical genomics study identifies Snf1 as a repressor of GCN4 translation. J Biol Chem 283:35889-98

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