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.
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.
