Dynamic post-translational modification (PTM) of histone proteins by chemical moieties such as acetyl-, methyl- and phospho- groups constitutes a key epigenetic mechanism that impacts on fundamental physiologic and pathologic processes. The proteins and domains that recognize distinct histone modification, named """"""""readers"""""""", define the functional consequences of specific modifications by transducing molecular events at chromatin to biological outcomes. Thus, the elucidation of histone PTM readers is critical for understanding how chromatin dynamics contribute to epigenetic programs, and how disruption of chromatin homeostasis fundamentally impacts on the development and progression of cancers and other diseases. The long-term goal of this proposal is to develop novel protein array technologies for proteome-wide, high- throughput discovery and study of the writers and readers of epigenetic events. A general hypothesis to be tested in this proposal is that protein array technology will revolutionize the ability of epigenetic researchers to discover diverse readers for the broad spectrum of chromatin PTMs present in the human epigenome. The proposal focuses on protein methylation as a prototypical epigenetic event, with two specific Aims planned. In the first Aim, paradigms will be established to validate and utilize multiplex modified histone peptide microarrays for high-throughput binding assays in screens employing protein domain libraries (developed in Aim 2), purified chromatin-regulatory macromolecular complexes and anti-histone antibodies. In chip enzymatic assays will be performed to investigate the utility of peptide microarrays as a platform for identifying candidate histone-modifying activities. In the second Aim, protein domain microarrays containing a comprehensive library of chromatin-associated domains will be generated and characterized as high-throughput tools for epigenetic research. Multiple types of probes will be developed for testing these arrays, including dual modification peptide probes (developed in Aim 1) and modification specific nucleosomal probes. For both Aims, analytic tools and software will be developed and standardized to facilitate transfer of the technology developed in the proposal to the epigenetic community. The generation in this proposal of new proteomic technologies that allow for rapid and accurate discovery of chromatin PTM readers should prove highly valuable for elucidating how epigenetic events influence human health and disease.

Public Health Relevance

Epigenetic events at chromatin regulate diverse cellular functions critical for normal health. Dysregulation of the proteins that place and read epigenetic modifications are frequently linked to neoplastic disease, immunodeficiencies syndromes and numerous other genetic disorders. Our proposed studies should provide insight into how epigenetic dynamics functional at the molecular level, and potentially lead to the discovery of new epigenetic targets for therapeutic intervention in diverse human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DA025800-02
Application #
7688691
Study Section
Special Emphasis Panel (ZRG1-CB-B (50))
Program Officer
Satterlee, John S
Project Start
2008-09-20
Project End
2011-01-31
Budget Start
2009-08-01
Budget End
2011-01-31
Support Year
2
Fiscal Year
2009
Total Cost
$285,995
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Calnan, Daniel R; Webb, Ashley E; White, Jamie L et al. (2012) Methylation by Set9 modulates FoxO3 stability and transcriptional activity. Aging (Albany NY) 4:462-79
Chang, Yanqi; Levy, Dan; Horton, John R et al. (2011) Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling. Nucleic Acids Res 39:6380-9
Woolard, Jeanette; Vousden, William; Moss, Steven J et al. (2011) Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms. Chem Sci 2011:273-278
Levy, Dan; Kuo, Alex J; Chang, Yanqi et al. (2011) Lysine methylation of the NF-?B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-?B signaling. Nat Immunol 12:29-36
Quinn, Amy M; Bedford, Mark T; Espejo, Alexsandra et al. (2010) A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics. Nucleic Acids Res 38:e11
Bua, Dennis J; Kuo, Alex J; Cheung, Peggie et al. (2009) Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks. PLoS One 4:e6789