Post-translational modifications of the N-terminal tail of histones play important roles in epigenetic regulation. The histone code hypothesis proposes that modifications of histone tails create combinatorial binding sites to recruit enzymes for transcriptional regulation. Dysregulation of histone modifications has been linked with diverse diseases including cancer. While much is known about histone modifications and the modifying enzymes, it remain largely unclear how the modified residues transmit the information to regulate gene expression. Identifications of modification-specific binding proteins will be the first step to fill this knowledge gap. Loss of acetylation of Lys16 (H4K16ac) and trimethylation of Lys20 of H4 (H4K20me3) has been correlated with cancer, but the molecular mechanism is not clear. The "binders" to H4K16ac and H4K20me3 are not known, hindering our understanding of how loss of these modifications may contribute to cancer. We propose to first develop a proteomic platform to identify specific histone modification binders and then use the platform to identify proteins that bind H4K16ac and H4K20me3. Our proteomics platform consists of efficient protein affinity purification with modified histone peptides with SILAC labeling and nanoHPLC-MS/MS for protein identification and quantification. We have preliminary data demonstrating the feasibility of such a platform using histone H3 peptides with modifications at Lys4 and/or Lys9. This Phase I proposal will allow us to: (1) develop a technology platform that will enable us to identify and quantify proteins that bind to any modifications on histone proteins; and (2) identify proteins that bind to H4K16ac and H3K20me3, that may have tumor suppressor functions. Our commercial activities will include: (1) contract services to academic and industry labs for the identification of histone code effectors; and (2) development of a Histone Code Reader Toolkit(r), which consists of recombinant proteins and antibodies that we find to bind modified histones to facilitate functional analysis of the histone code in epigenetics field.
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