Bromodomain-PHD Finger protein 1 (BRPF1) is a key regulatory subunit of the MOZ/MORF histone acetyltransferase (HAT) complex that contains a unique assembly of two Plant Homeodomain (PHD) fingers separated by a zinc knuckle, designated PZP (PHD1-Zn-PHD2). PHD fingers are important protein modules found in a variety of cardiac gene expression regulators, whose disruption effects cardiac muscle fiber development and leads to heart disease. Recently, we found that PHD1 of BRPF1 binds to the histone H3 tail. However, the structure and function of the BRPF1 PHD fingers are not known, and the PZP assembly appears to be a novel chromatin binding module who's PHD fingers may act in concert to confer additional outcomes not seen with single PHD domains. This proposal aims to determine how binding of the PZP assembly targets BRPF1 to the histone tail using structural and functional techniques. We will identify the PHD1, PHD2 and PZP histone tail ligand(s) and investigate the effects of post-translational modifications on binding using western-blot pull down assays. The structures of PHD1 and PHD2 in complex with their histone tail ligand will be solved using NMR or X-ray crystallographic techniques to investigate the molecular basis for the recognition of unmodified/modified histones. This will allow us to analyze specific interactions of these chromatin binding modules with histone peptides at the atomic level. Once we have interpreted the structural data, we will use this information to make mutations in PHD1, PHD2 and PZP to test which residues are most important for the interaction with the histone tail. We will measure the effect of these mutations on histone binding using NMR, tryptophan fluorescence, or ITC by titrating in histone peptides to determine their dissociation constants (Kds). To establish the co-active or competitive nature of the wild-type PHD1-Zn-PHD2 domains we will compare the Kds of individual PHD fingers to the whole PZP assembly. The functional significance of wild-type and mutant proteins will also be tested in vitro using HAT assays, and in vivo by chromatin immunoprecipitation and fluorescence microscopy. These data will provide critical structural insights into the molecular mechanism used by the BRPF1 PZP assembly to target the histone tail, and will impart a greater understanding on how this novel chromatin binding effector links epigenetic regulation via PHD domains to the regulation of pathological cardiac gene expression.
|Program type||Beginning Grant-in-Aid|
|Effective start/end date||07/01/2010 → 06/30/2012|