This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technology Core Projects 5: This core technology involves the use of chemical affinity labeling reagents to discover and characterize novel histone acetyltransferase (HAT) enzymes. It is hypothesized here that families of HATs remain to be discovered, in part due to the limited approaches that have been so far employed for HAT identification. By applying active site labeling agents, it should be possible to find new HAT enzymes which can open new vistas in our understanding of gene regulation.
Specific Aim 1. Synthesize a series of chemically reactive CoA analogs for affinity labeling studies. CoASH, generated in radiolabeled form containing 3''''''''''''''''''''''''''''''''-32p (or 3''''''''''''''''''''''''''''''''-33p), will be used as a precursor to synthesize a series of intrinsically reactive or photoreactive reagents. The target compounds will be varied in terms of the distance between the electrophilic/photoactive moiety from the CoA core and the degree of reactivity toward nucleophilic or non-nucleophilic enzyme groups.
Specific Aim 2. Evaluate the CoA affinity reagents with known, purified HATs, and spiked HATs in mixtures and immobilized in microarrays.. The CoA affinity reagents will be tested as enzyme inhibitors individually with purified p300, PCAF, EsaI, and serotonin N-acetyltransferase to assess active site interactions. Based on these studies, crosslinking experiments with suitable ranges of compound concentration, buffer pH, and reaction times will be performed. To assess specificity, crosslinking experiments in the absence and presence of competing desulfoCoA will be carried out. Stoichiometry of labeling will be determined by scintillation counting and/or phosphorimager analysis. After optimizing conditions with purified proteins, compounds will be employed in cell extracts spiked with mixtures to determine the level of specificity that can be achieved in a more practical setting. In collaboration with Heng Zhu, they will also be examined on glass slide immobilized HATs.
Specific Aim 3. Identify and characterize novel CoA-crosslinked proteins as potential HATs. A subset of compounds culled from experiments in Specific Aim 2 will be tested to identify unknown bands in extracts and with spatially separated proteomes on slides. Cell extracts will be separated by 2D-gel electrophoresis and visualized by phosphorimage analysis. Bands corresponding to labeled proteins from extracts will be isolated and identified by modern mass spec methods in collaboration with Bob Cotter. Proteins from extracts as well as from protein chips, judged to be most interesting based on their DNA sequences based on consultation with our Co-PIs Jef Boeke and Shelly Berger, will be expressed and assayed for HAT activity. Promising enzymes will be characterized more deeply in cellular stu
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