Protein lysine acetyltransferases are key enzymes in determination of cell fate though regulation of chromatin structure, gene expression, protein stability, and complex formation. The homologues p300 and CBP are perhaps the most renowned acetyltransferases due to their implication in a diversity of diseases including many developmental disorders and cancers. Although many deacetylase drugs have been developed, only very recently was the first acetyltransferase inhibitor patented, specifically for p300 and CBP. However little about this inhibitor's effects on genetic expression and the chromatin landscape is known. Coupled with recent data showing activation of p300/CBP nucleosome acetylation by an allosteric bromodomain ligand and concomitant significant inhibition of cellular proliferation, these ligands are primary tools to address the function and interplay between the ?writer? acetyltransferase domain and the adjacent ?reader? bromodomain. Previous and preliminary data corroborate the hypothesis of potential crosstalk between the domains. Therefore, the goals of the current proposal commencing in the K99 phase are to 1) Delineate the multifaceted regulation of the p300/CBP bromodomain and HAT domain in governing protein acetyltransferase function, specificity, and the genetic landscape using cellular drug co-treatment strategies and in vitro biochemistry; and 2) Identify novel protein substrates whose acetylation can be specifically modulated by p300/CBP ligand(s) by protein microarrays and characterize these acetylated substrates by expressed protein ligation followed by protein biochemistry/ molecular biology. Using the techniques and skills mastered in this phase, the R00 phase will apply these to the neglected yet unarguably pivotal MYST-family acetyltransferase MOZ. This will include 3) identifying the specific acetylation sites on nucleosomes and other protein substrates catalyzed by MOZ, and characterizing the function of these modification(s) on substrate protein function. The K99 training phase is transferred to Brigham and Women's Hospital. The trainee comes from a background in biochemistry and molecular biology. This training will build skills in pharmacology, chemical biology, and high-throughput epigenetic landscape analyses necessary to commence an independent career in epigenetic biochemistry. This includes but is not limited to post-translational modification site identification by mass spectrometry, expressed protein ligation, ChIP-Seq/RNA-Seq bioinformatics, and drug synergy studies. Courses in the respective disciplines will be audited. Professionally, the applicant will be taught strategies in academic laboratory leadership, mentoring and pedagogy. Upon completion of this training, the applicant will be more adequately equipped to commence on an academic research career focusing on the substrate selectivity and regulation of epigenetic enzymes. Members of the applicant's advisory committee will provide scientific and professional advice throughout all phases of the proposal.
The goal of this proposal is to identify critical nodes of regulation of the lysine acetyltransferase enzymes p300/CBP and MOZ which are commonly misregulated in a diversity of human diseases. This will include identifying and characterizing the function of novel protein substrates of these enzymes and probing the effect of enzyme active-site and allosteric ligands on chromatin structure and subsequent genetic expression. This work is fundamental to designing disease- specific therapies for the plethora of aberrant activities of these enzymes across a spectrum of disorders.
|Weinert, Brian T; Narita, Takeo; Satpathy, Shankha et al. (2018) Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell 174:231-244.e12|
|Zucconi, Beth E; Cole, Philip A (2017) Allosteric regulation of epigenetic modifying enzymes. Curr Opin Chem Biol 39:109-115|