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 nucleosomes comprising H3K27me3. 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 NPM1 interacts directly with PRC2. Notably, the NPM1 mutant (NPM1c) associated with ~35% of all Acute Myelogenous Leukemia (AML) is mis-localized to the cytoplasm. 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 vivo. We will further 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. With a different perspective, we will follow the interactive dynamics of NPM1 and PRC2 in the context of a replication fork using single-molecule localization microscopy as well as stochastic optical reconstruction microscopy (STORM). These approaches should bear directly on the role of NPM1 as an ?epigenetic histone chaperone? and the significance of its interaction with the epigenetic regulator, PRC2. Importantly, we propose that the NPM1c mutant associated with AML results in a mis-regulated PRC2. Indeed, known HOX gene targets of PRC2 are aberrantly expressed in NPM1c AML, and participate in the establishment of the leukemic state. While deposition of H3K27me3 by PRC2 and DNA methylation by DNMT3A result in repressed chromatin, these events 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?a compensation that is lost upon DNMT3A mutation. We are in position to fully grasp the sequence of aberrant epigenetic events as they occur during leukemogeneis. We will track the repercussions to gene expression, features of repressed chromatin domains, PRC2 chromatin occupancy and DNA methylation in both tissue culture and a mouse model of AML, as NPM1c is temporally expressed as a function of the presence of mutant DNMT3A. These results will pinpoint the initial, aberrant epigenetic events giving rise to AML. During the Bridging funding, we will concentrate on the in vivo cell based assay that we developed to precisely define the function of NPM1 during mitotic inheritance of H3K27me2/me3-repressive chromatin domains, as described in the Experimental section, below.

Public Health Relevance

The inheritance of a particular gene expression profile is key to conveying proper cellular identity to progeny cells upon cell division. The proposed studies will directly expose key mechanistic aspects and biologically relevant factors inherent to this epigenetic regulation, as well as the sequence of events that accompany leukemogenesis as a consequence of naturally occurring mutations that target this inheritance.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA199652-17S2
Application #
10225025
Study Section
Program Officer
Mietz, Judy
Project Start
2020-08-01
Project End
2021-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
17
Fiscal Year
2020
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Stafford, James M; Lee, Chul-Hwan; Voigt, Philipp et al. (2018) Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma. Sci Adv 4:eaau5935
Lee, Chul-Hwan; Holder, Marlene; Grau, Daniel et al. (2018) Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2. Mol Cell 70:435-448.e5
Lee, Chul-Hwan; Yu, Jia-Ray; Kumar, Sunil et al. (2018) Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin. Mol Cell 70:422-434.e6
Krishnan, Swathi; Smits, Arne H; Vermeulen, Michiel et al. (2017) Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis. Mol Cell 67:579-593.e6
Narendra, Varun; Bulaji?, Milica; Dekker, Job et al. (2016) CTCF-mediated topological boundaries during development foster appropriate gene regulation. Genes Dev 30:2657-2662
Justin, Neil; Zhang, Ying; Tarricone, Cataldo et al. (2016) Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat Commun 7:11316
Campos, Eric I; Smits, Arne H; Kang, Young-Hoon et al. (2015) Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event. Mol Cell 60:697-709