Regulatory proteins serve as integrators of inter-and intracellular signals. Cellular information is often communicated in a language of posttranslational protein modification. This theme has been particularly apparent in cellular signaling pathways that impinge on the transcriptional program coordinated in the nucleus of the cell. Protein acetylation has been known for many years, but our knowledge of proteins regulated by acetylation is considerably less than for phosphorylation. Recent studies indicate that several key transcriptional regulators including; HNF-4 and NFkappaB are regulated by acetylation, which underscores the need to better understand the relevant acetylation pathways that govern the function of these molecules. Dissecting these regulatory circuits will hopefully lead to a better understanding and treatment of diabetes, cancers and other human diseases. This proposal describes a chemical genetic approach to the elucidation of histone acetyltransferase function in vivo. This technique will provide two types of information that will be invaluable in dissecting HAT signaling pathways: (1) to directly and unambiguously identify the complete set of in vivo acetyltransferase substrate molecules for two histone acetyltransferase (HAT) enzymes, Gcn5 and PCAF, and (2) to define the downstream affects of these acetylation signaling pathways in vivo, using chromatin immunoprecipitation assays (CHIP). During the training period covered by this award, I will focus on developing the necessary skills to transition into an independent investigator at the interface of the chemical and biological sciences. This will require that I gain experience with new techniques including computer-based molecular modeling, proteimic based protein isolation and identification techniques (including the mass spectrometric microsequencing of cellular proteins) and cell biological techniques including chromatin immunoprecipitation and immunofluorescence microscopy.
