Each somatic cell in a human body contains the same genetic information, but the diverse function of differentiated cells is made possible through precise control of gene activity. Proper gene regulation is crucial for the normal function of a cell, and human diseases can and do result from loss or circumvention of normal gene regulation. Our long term objective is therefore to understand how gene expression is regulated in eukaryotic cells. This grant proposal focuses on transcriptional coactivators which enable the TATA-binding protein (TBP) to bind to its TATA-box DMA binding site, a critical step in initiating gene transcription. Significant data indicates that the SAGA histone acetyltransferase (HAT) complex functions as a coactivator by delivering TBP to the promoter and by acetylating nucleosomes in the promoter region. Despite major advances in the last decade, the molecular mechanism for either activity is not well-understood. For example, we do not know precisely how SAGA recruits TBP to the core promoter. Similarly, while we do understand how HAT subunits recognize histone tail substrates, we do not have an equivalent understanding of how HAT or other chromatin enzymes recognize their physiological nucleosome substrate.
Our specific aims are directed to address these deficiencies. In particular, we propose to: 1. Determine how SAGA's Spt8 subunit binds to TBP. We will perform mutagenesis and label transfer experiments to define the TBP and Spt8 surfaces that interact. 2. Determine how SAGA recruits TBP to a promoter TATA-box. We will develop and utilize in vitro assays to examine the molecular mechanism for how SAGA delivers TBP to a promoter. 3. Determine how the Ada2/Ada3/Gcn5 and Piccolo/NuA4 HAT enzymes interact with nucleosomes. We will use photoaffinity labeling to identify HAT complex subunits in close proximity to nucleosomes, and deletion and mutagenesis studies to define novel functions for the bromo and chromodomains in the binding and acetylation of nucleosomes. Our biochemical studies using defined components will complement the considerable available in vivo data by addressing mechanistic questions not easily tackled by other approaches.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Preusch, Peter C
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Jennings, Matthew J; Barrios, Adam F; Tan, Song (2016) Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site. Protein Expr Purif 121:17-21
McGinty, Robert K; Tan, Song (2015) Nucleosome structure and function. Chem Rev 115:2255-73
McGinty, Robert K; Henrici, Ryan C; Tan, Song (2014) Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514:591-6
Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C et al. (2013) Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity. Genes Dev 27:2009-24
Huang, Jiehuan; Tan, Song (2013) Piccolo NuA4-catalyzed acetylation of nucleosomal histones: critical roles of an Esa1 Tudor/chromo barrel loop and an Epl1 enhancer of polycomb A (EPcA) basic region. Mol Cell Biol 33:159-69
Tan, Song (2012) Deciphering how the chromatin factor RCC1 recognizes the nucleosome: the importance of individuals in the scientific discovery process. Biochem Soc Trans 40:351-6
Chatterjee, Nilanjana; Sinha, Divya; Lemma-Dechassa, Mekonnen et al. (2011) Histone H3 tail acetylation modulates ATP-dependent remodeling through multiple mechanisms. Nucleic Acids Res 39:8378-91
Charles, Georgette M; Chen, Changbin; Shih, Susan C et al. (2011) Site-specific acetylation mark on an essential chromatin-remodeling complex promotes resistance to replication stress. Proc Natl Acad Sci U S A 108:10620-5
Chittuluru, Johnathan R; Chaban, Yuriy; Monnet-Saksouk, Julie et al. (2011) Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes. Nat Struct Mol Biol 18:1196-203
Tan, Song; Davey, Curt A (2011) Nucleosome structural studies. Curr Opin Struct Biol 21:128-36

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