The long-term goal of our labs is to determine the molecular mechanisms by which post-translational modifications of histones regulate gene expression and DNA repair. These two cellular processes are vital for the growth and health of all organisms. Alterations in post-translational histone modifications affect the regulation of gene expression and DNA repair and can lead to diseases such as ICF, Rett and ATRX syndromes and, most predominantly, cancer. This research project will determine the molecular mechanisms by which four key post-translational modifications at histone H3 residues K56, K115, T118 and K122 function. These modifications were recently identified in structured regions of the nucleosome. They occur both individually and together in vivo, and are critical for transcriptional regulation and DNA repair. The mechanisms by which these modifications carry out vital biological functions remain poorly understood. However, each modification is buried beneath DNA in the histone-DNA interface at one of two critical regions of the nucleosome: the dyad symmetry axis and the DNA entry-exit region. This implies that the mechanisms by which these modifications function must require significant changes in nucleosome conformation and/or dynamics. Therefore, combined biochemical and biophysical studies are key to understanding the role of these modifications. The four modifications will be studied by reconstituting uniformly modified semi-synthetic nucleosomes using histone proteins that are constructed by expressed protein ligation and by sequential chemical ligation. Multiple approaches will be used to quantify modification-induced changes in chromatin conformation and dynamics: fluorescence resonance energy transfer, restriction enzyme digestions, nucleosome mapping, nucleosome competitive reconstitutions, stopped flow fluorometry and fluorescence correlation spectroscopy. These experiments will determine whether and how these four histone H3 modifications in the DNA-histone interface regulate, in Aim 1, Nucleosome Structure and DNA site Exposure, in Aim 2, Nucleosome Positioning and Stability and, in Aim 3, Nucleosome Dynamics. The successful completion of this research project will make a major impact on scientific knowledge and the molecular understanding of disease in two ways. First, it will determine how four modifications in histone H3 buried within the nucleosome DNA-histone interface facilitate both DNA repair and transcriptional regulation. Second, it will provide insight into the possible mechanisms of over 20 post-translational histone modifications that are known to be buried throughout the nucleosome DNA-histone interface.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083055-05
Application #
8215741
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2008-02-01
Project End
2013-08-31
Budget Start
2012-02-01
Budget End
2013-08-31
Support Year
5
Fiscal Year
2012
Total Cost
$279,329
Indirect Cost
$93,110
Name
Ohio State University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
North, Justin A; Šimon, Marek; Ferdinand, Michelle B et al. (2014) Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure. Nucleic Acids Res 42:4922-33
Luo, Yi; North, Justin A; Rose, Sean D et al. (2014) Nucleosomes accelerate transcription factor dissociation. Nucleic Acids Res 42:3017-27
North, Justin A; Amunugama, Ravindra; Klajner, Marcelina et al. (2013) ATP-dependent nucleosome unwrapping catalyzed by human RAD51. Nucleic Acids Res 41:7302-12
Sen, Payel; Vivas, Paula; Dechassa, Mekonnen Lemma et al. (2013) The SnAC domain of SWI/SNF is a histone anchor required for remodeling. Mol Cell Biol 33:360-70
Gao, Min; Nadaud, Philippe S; Bernier, Morgan W et al. (2013) Histone H3 and H4 N-terminal tails in nucleosome arrays at cellular concentrations probed by magic angle spinning NMR spectroscopy. J Am Chem Soc 135:15278-81
Law, Yu Kay; Forties, Robert A; Liu, Xin et al. (2013) Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA. Photochem Photobiol Sci 12:1431-9
Shimko, John C; Howard, Cecil J; Poirier, Michael G et al. (2013) Preparing semisynthetic and fully synthetic histones h3 and h4 to modify the nucleosome core. Methods Mol Biol 981:177-92
Kodgire, Prashant; Mukkawar, Priyanka; North, Justin A et al. (2012) Nucleosome stability dramatically impacts the targeting of somatic hypermutation. Mol Cell Biol 32:2030-40
North, Justin A; Javaid, Sarah; Ferdinand, Michelle B et al. (2011) Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling. Nucleic Acids Res 39:6465-74
Forties, Robert A; North, Justin A; Javaid, Sarah et al. (2011) A quantitative model of nucleosome dynamics. Nucleic Acids Res 39:8306-13

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