Our lab is interested in the biochemical mechanisms by which the RNA polymerase II preinitiation complex (PIC) coordinates its functions with histone modification and remodeling enzymes necessary for transcription on chromatin. Work in previous funding cycles has established models for how the Mediator and TFIID co-activators coordinate with p300, Chd1, and SAGA, and how silencing proteins like HP1 and PRC1 influence this process on designer chromatin templates bearing specific histone modifications. Our most recent experiments have focused on how the H2AZ-H3.3 variant chromatin found at eukaryotic promoters regulates transcription. We have for the first time recreated transcriptional stimulation by acetylated variant chromatin in vitro, shown that it enhances binding of p400-Tip60 and Ino80, and will now pursue its mechanism. We employ the immobilized template assay, which allows us to capture PICs from HeLa and mouse embryonic stem cell extracts and analyze the functions of different PIC factors in vitro. The in vitro studies are accompanied by ChIP in a model cell-based system, and by analysis of genome wide data sets in the gene expression omnibus.
In Aim 1, we will acquire knowledge of the role of enhanced binding of p400-Tip60 to variant chromatin on gene activation.
In Aim 2, we will analyze binding and function of Ino80 on variant chromatin and the role Mediator plays in this process. Our emphasis is on how Ino80 responds to nucleosomal obstacles containing and lacking H2AZ and H3.3.
Aim 3 examines the specific subunits dictating interaction of Mediator with TFIID, which form the core structure that controls binding of general factors, Pol II and the numerous chromatin factors necessary for initiation. Our results will provide a detailed mechanism of gene activation on chromatin using state of the art biochemical approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM074701-09A1
Application #
8696675
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2005-08-01
Project End
2018-01-31
Budget Start
2014-05-01
Budget End
2015-01-31
Support Year
9
Fiscal Year
2014
Total Cost
$346,500
Indirect Cost
$121,500
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Pradhan, Suman K; Su, Trent; Yen, Linda et al. (2016) EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation. Mol Cell 61:27-38
Xue, Yong; Van, Christopher; Pradhan, Suman K et al. (2015) The Ino80 complex prevents invasion of euchromatin into silent chromatin. Genes Dev 29:350-5
Sridharan, Rupa; Gonzales-Cope, Michelle; Chronis, Constantinos et al. (2013) Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency. Nat Cell Biol 15:872-82
Chen, Xiao-Fen; Lehmann, Lynn; Lin, Justin J et al. (2012) Mediator and SAGA have distinct roles in Pol II preinitiation complex assembly and function. Cell Rep 2:1061-7
Lehmann, Lynn; Ferrari, Roberto; Vashisht, Ajay A et al. (2012) Polycomb repressive complex 1 (PRC1) disassembles RNA polymerase II preinitiation complexes. J Biol Chem 287:35784-94
Lin, Justin J; Carey, Michael (2012) In vitro transcription and immobilized template analysis of preinitiation complexes. Curr Protoc Mol Biol Chapter 12:Unit 12.14.
Kitada, Tasuku; Kuryan, Benjamin G; Tran, Nancy Nga Huynh et al. (2012) Mechanism for epigenetic variegation of gene expression at yeast telomeric heterochromatin. Genes Dev 26:2443-55
Kuryan, Benjamin G; Kim, Jessica; Tran, Nancy Nga H et al. (2012) Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro. Proc Natl Acad Sci U S A 109:1931-6
Lin, Justin J; Lehmann, Lynn W; Bonora, Giancarlo et al. (2011) Mediator coordinates PIC assembly with recruitment of CHD1. Genes Dev 25:2198-209
Smallwood, Andrea; Black, Joshua C; Tanese, Naoko et al. (2008) HP1-mediated silencing targets Pol II coactivator complexes. Nat Struct Mol Biol 15:318-20

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