LELAND STANFORD JUNIOR UNIVERSITY, THE
The PHD finger is a signature chromatin-associated protein motif, mutations in which are associated with cancers, immunodeficiency syndromes, and other genetic disorders. Our long-term goal is to develop a comprehensive molecular understanding of how PHD domains impact on chromatin dynamics and the relationship of such activities to fundamental nuclear functions and human disease processes. The general hypothesis to be tested in this proposal is that PHD domains recognize specific methylated histone marks. Preliminary work indicates that several PHD domains bind with high affinity and specificity to histone H3 methylated at lysine 4 (H3K4me3). Characterization of the physiologic role of H3K4me3-recognition by model PHD domains should be instrumental for elucidating how disruption in chromatin dynamics can contribute to numerous pathologic states. Here, a series of biochemical, cellular, and proteomic analyses are proposed to investigate the molecular activities of PHD domains. (1) To determine the molecular function of the ING2 PHD domain in H3K4me recognition at chromatin. Biochemical and functional studies are proposed to investigate the hypothesis that the PHD domain of the ING2 tumor suppressor protein is a specific effector molecule of H3K4me3. We will (i) elucidate the molecular basis of ING2 PHD domain specificity for H3K4me states, (ii) determine how H3K4me impacts on ING2-associated histone deacetylase activity, and (iii) characterize regulation of the ING2-H3K4me3 interaction by phosphoinositides. (2) To elucidate the cellular functions of H3K4me recognition by the ING2 PHD domain. Based on preliminary work, we will investigate the hypothesis that the ING2 PHD domain links H3K4me3 to acute gene repression. We will characterize the physiologic interaction of the ING2 PHD domain with H3K4me at target genes, and determine how these interactions impact on gene expression programs. We will also test how select nuclear factors regulate ING2-H3K4me functions. (3) To characterize the H3K4me-recognition activity of specific PHD fingers with functions distinct from ING2. Preliminary work indicates that besides ING2, the PHD domains of the other ING proteins and that of the RAG2 recombinase bind H3K4me3, potentially linking H3K4me3 to diverse nuclear processes. Biochemical and functional approaches are proposed to study the biology of H3K4me3-recognition by these PHD fingers-containing proteins.