Project Summary A critical question in the field of Epigenetics/Mammalian Gene Regulation is how a cellular identity is inherited by progeny cells during cell division. This fundamental aspect of epigenetic regulation was recently clarified in our lab: repressed, but not active, chromatin domains are inherited. Repressed chromatin domains in facultative heterochromatin are maintained by the multi-subunit complex, Polycomb Repressive Complex 2 (PRC2), that catalyzes the histone post-translational modification, H3K27me3. PRC2 exhibits a notable “read and write” feature whereby its recognition of H3K27me3 results in its allosteric activation. Thus, PRC2 can fully restore repressive chromatin domains upon inheritance of H3K27me3-nucleosomes. Remarkably, our findings point to a previously reported histone chaperone, NPM1, as facilitating this inheritance of repressed chromatin: NPM1 is exclusively localized to chromatin in late S-phase when repressed chromatin is replicated, and interacts directly with PRC2. Our latest findings demonstrated specific de-repression of PRC2-regulated genes upon auxin- mediated depletion of NPM1 during S-phase of the cell cycle. We will expand our mechanistic studies of epigenetic inheritance by investigating the role of NPM1 as an S-phase-specific histone chaperone and its interplay with PRC2 in a series of histone chaperone assays performed in vitro with distinct candidate oligonucleosomal templates. We will investigate the role of NPM1 in epigenetic inheritance by adapting our in vivo assay for chromatin domain inheritance as a function of the presence of NPM1 and pertinent NPM1 mutants. The interactive dynamics of NPM1 and PRC2 in the context of a replication fork is critical information towards understanding the transfer of parental nucleosomes to daughter DNA strands. Thus, single-molecule localization microscopy as well as stochastic optical reconstruction microscopy (STORM) are expected to bear directly on the role of NPM1 and the significance of its interaction with the epigenetic regulator, PRC2. Importantly, mutant NPM1c associated with ~35% of all Acute Myelogenous Leukemia (AML) is mis-localized to the cytoplasm. We propose that NPM1c hampers normal PRC2 function. Indeed, similar to our findings above upon NPM1 depletion, known HOX gene targets of PRC2 are aberrantly expressed in NPM1c AML, participating in the establishment of the leukemic state. Deposition of H3K27me3 by PRC2 and DNA methylation by DNMT3A result in repressed chromatin, but are usually mutually exclusive. Yet, NPM1 and DNMT3A mutations synergize in leukemogenesis. Thus, we further propose that DNMT3A partially compensates for our proposed NPM1c- mediated thwarting of PRC2⏤which is lost upon DNMT3A mutation. Through temporal expression of NPM1c as a function of the presence of mutant DNMT3A, we will track the repercussions to gene expression, features of repressed chromatin domains, PRC2 chromatin occupancy and DNA methylation in both tissue ...