The Inhibitor of Growth 1 (ING1) tumor suppressor has been implicated in vital cellular processes including cell cycle regulation, cellular senescence, apoptosis and DNA repair. ING1 is involved in regulation of chromatin structure and gene expression; however, its role in these processes remains unclear. The plant homeodomain (PHD) finger of ING1 recognizes tri-methylated lysine 4 of histone H3 (H3K4Me3) a histone mark associated with active chromatin and gene transcription. The N-terminal SAID domain of ING1 associates with the SAP30 component of the mSin3A-HDAC1/2 repressive complex. Mutations in the PHD finger and in the SAID domain are found in several cancers; however the molecular mechanisms underlying these interactions are not determined. I hypothesize that ING1 anchors the mSin3A-HDAC1/2 complex to chromatin through interactions of its SAID domain and PHD finger with SAP30 and H3K4Me3, respectively. An understanding of these interactions at the molecular level will help uncover the mechanism through which ING1 exerts its function as a tumor suppressor. To define the molecular basis of H3K4Me3 recognition by ING1 PHD finger and of ING1 SAID domain association with SAP30, we will determine the atomic resolution structures of these modules by NMR spectroscopy or X-ray crystallography. The H3K4Me3 binding pocket will be defined from the structure of the PHD-H3K4Me3 complex, whereas the SAP30 binding site will be identified by chemical shift changes in the SAID domain induced by SAP30. In both domains, the binding site residues and residues known to be mutated in cancers will be substituted and the binding energetics will be analyzed. We will further analyze the interactions by western analysis and fluorescence microscopy of green-fluorescent protein-tagged ING1. Next, the mutant full-length ING1 proteins will be used in chromatin immunoprecipitation assays to test whether the SAID domain and PHD finger are necessary for the recruitment of the mSin3A/HDAC1/2 complex to H3K4Me3. These studies will help to better understand the tumor suppressive function of ING1, which is present at lower levels in cancer cells than in normal cells. By studying the 3D structure of ING1, we will elucidate its mechanism of action at the atomic level. Because ING1 associates with enzymes that are targets of cancer drugs currently in clinical trials, ING1 may be a novel target for design of novel therapeutic agents. ? ? ?
