Our long-term objectives are to elucidate fundamental aspects about transcriptional activation in the context of chromatin. A variety of multiprotein coactivators, ATP-dependent (e.g., Swi/Snf) chromatin remodelers and histone acetyltransferases (e.g., SAGA and NuA4), enable the transcriptional apparatus to contend with repressive chromatin structures in eukaryotes. Hence, they are critical to the proper regulation of transcription by RNA polymerase II. This proposal builds on novel molecular approaches that we have developed to increase our understanding of in vivo transcriptional activation mechanisms, using the genetically tractable organism, S. cerevisiae, as a model system. The yeast promoters PH05 and PH08, while coordinately regulated by the same phosphate signaling pathway, have different requirements for transcriptional coactivators. We propose to investigate the molecular basis for these differences by determining how the phenotypic consequences of disruption of coactivator functions varies as a function of promoter occupancy by Pho4. We will also ascertain the extent to which cooperative DNA binding by Pho4 and Pho2, or a functional interaction of their activation domains, leads to additive or synergistic increases in transcription, thereby altering the necessity for individual coactivators at PH05 or PH08. In addition, we will examine whether increased activator binding leads to recruitment of 'excess' chromatin remodelers, establishing functional redundancy, where activation is refractory to inactivation of individual complexes. These studies will utilize chromatin immunoprecipitation and a powerful strategy, TAGM, for quantitative determination of relative activator and coactivator binding in intact cells. We also propose to characterize whether the disruption of multiple nucleosomes at the PH05 and PH08 promoters is sequential, occurring in a random or defined order, or is cooperative. These studies will utilize a novel single-molecule application of our previously published approaches for monitoring chromatin remodeling in vivo with DNA MTases. The studies should lead to important insights into the regulation of transcription. They also have relevance to human health as recent discoveries implicate chromatin and its constituents in cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA095525-05
Application #
7224961
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Couch, Jennifer A
Project Start
2003-05-01
Project End
2010-04-30
Budget Start
2007-05-01
Budget End
2010-04-30
Support Year
5
Fiscal Year
2007
Total Cost
$247,257
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Darst, Russell P; Haecker, Irina; Pardo, Carolina E et al. (2013) Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus. Nucleic Acids Res 41:2993-3009
Darst, Russell P; Nabilsi, Nancy H; Pardo, Carolina E et al. (2012) DNA methyltransferase accessibility protocol for individual templates by deep sequencing. Methods Enzymol 513:185-204
Darst, Russell P; Pardo, Carolina E; Pondugula, Santhi et al. (2012) Simultaneous single-molecule detection of endogenous C-5 DNA methylation and chromatin accessibility using MAPit. Methods Mol Biol 833:125-41
Pardo, Carolina E; Carr, Ian M; Hoffman, Christopher J et al. (2011) MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects. Nucleic Acids Res 39:e5
Pardo, Carolina E; Darst, Russell P; Nabilsi, Nancy H et al. (2011) Simultaneous single-molecule mapping of protein-DNA interactions and DNA methylation by MAPit. Curr Protoc Mol Biol Chapter 21:Unit 21.22
Darst, Russell P; Pardo, Carolina E; Ai, Lingbao et al. (2010) Bisulfite sequencing of DNA. Curr Protoc Mol Biol Chapter 7:Unit 7.9.1-17
Dechassa, Mekonnen Lemma; Sabri, Abdellah; Pondugula, Santhi et al. (2010) SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes. Mol Cell 38:590-602
Pondugula, Santhi; Neef, Daniel W; Voth, Warren P et al. (2009) Coupling phosphate homeostasis to cell cycle-specific transcription: mitotic activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead proteins. Mol Cell Biol 29:4891-905
Pardo, Carolina; Hoose, Scott A; Pondugula, Santhi et al. (2009) DNA methyltransferase probing of chromatin structure within populations and on single molecules. Methods Mol Biol 523:41-65
Pondugula, Santhi; Kladde, Michael P (2008) Single-molecule analysis of chromatin: changing the view of genomes one molecule at a time. J Cell Biochem 105:330-7

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