The formation of RNA polymerase II (Pol II) preinitiation complexes (PICs) on chromatin is one of the most fundamental processes in eukaryotic gene regulation. Genetics, chromatin immunoprecipitation and molecular studies in mammalian cells have provided numerous insights into PIC assembly on chromatin in vivo. Despite the extensive literature, there is a poor understanding of the actual biochemical mechanisms. Few labs have studied the problem using systems that allow analysis of the effect of chromatin modifications on PICs in vitro. The opposing phenomena of histone H3K4 and H3K27 methylation mediated by the trithorax (MLL/Set1) and polycomb (PRC2) complexes, respectively, are of particular interest due to the important role they play during development and differentiation. A rudimentary understanding of their mechanism requires that these events be recreated and analyzed in a defined transcription system. There has been much work done on the basic enzymology of the PRC1, PRC2 and MLL/Set1 complexes. I propose to significantly extend these studies to address important aspects of how these complexes affect PIC assembly in both our model GAL4-VP16 in vitro system and on natural stem cell promoters controlled by Sox2, Oct4, Myc, Klf4 and Nanog. A particularly powerful technique that will form the cornerstone of the proposal is the immobilized template assay. This assay employs biotinylated templates assembled into chromatin with naove and chemically methylated histones. The templates are attached to streptavidin-coated beads to capture the PICs from extracts and analyze their composition by immunoblotting. The functions of the PIC are studied using histone modification/remodeling assays and in vitro transcription. The immobilized template approach will be used to address the following three aims:
Aim #1 will examine the how MLL/Set1 and H3K4 methylation affect the assembly of a PIC.
Aim #2 will examine the specific mechanism of silencing by PRC1 and PRC2 on the PIC and the mechanism of bivalent domains containing both trimethylated H3K4 and H3K27.
Aim #3 will examine how stem cell activators assemble into enhanceosomes and activate transcription of chromatinized Sox2 and Nanog genes in vitro. Our study will leverage the vast body of knowledge from mammalian gene regulation to craft and test hypotheses for how PICs assemble on nucleosomes and how covalent modifications of chromatin, and the machines that bind those modifications, regulate this process. The knowledge will provide fundamental information applicable to transcription regulation in many organisms.

Public Health Relevance

One of the most important aspects of gene regulation is understanding how chromatin, the nucleoprotein structure that protects eukaryotic genome, controls when genes are turned on and replaced when genes are turned off. This step is fundamental to all organisms and knowledge of the process is key to understanding gene regulation during disease, differentiation and development in humans. Our proposal will use a model system to understand the biochemical details and mechanism of this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074701-08
Application #
8301629
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2005-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
8
Fiscal Year
2012
Total Cost
$316,024
Indirect Cost
$105,302
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
Sridharan, Rupa; Gonzales-Cope, Michelle; Chronis, Constantinos et al. (2013) Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1? in reprogramming to pluripotency. Nat Cell Biol 15:872-82
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; Carey, Michael (2012) In vitro transcription and immobilized template analysis of preinitiation complexes. Curr Protoc Mol Biol Chapter 12:Unit 12.14.
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
Black, Joshua C; Mosley, Amber; Kitada, Tasuku et al. (2008) The SIRT2 deacetylase regulates autoacetylation of p300. Mol Cell 32:449-55
Smallwood, Andrea; Esteve, Pierre-Olivier; Pradhan, Sriharsa et al. (2007) Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes Dev 21:1169-78
Black, Joshua C; Choi, Janet E; Lombardo, Sarah R et al. (2006) A mechanism for coordinating chromatin modification and preinitiation complex assembly. Mol Cell 23:809-18
Carey, Michael; Li, Bing; Workman, Jerry L (2006) RSC exploits histone acetylation to abrogate the nucleosomal block to RNA polymerase II elongation. Mol Cell 24:481-7