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.
|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|