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Principal Investigator: DANNY REINBERG
Organization: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
Fiscal Year: 2024
Award: $346,334
Funding agency: National Cancer Institute
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 culture and a
mouse model of AML to fully grasp the sequence of aberrant epigenetic events as they occur in leukemogenesis.
Terms: <AML - Acute Myeloid Leukemia><Acute Myeloblastic Leukemia><Acute Myelocytic Leukemia><Acute Myelogenous Leukemia><Adult AGL><Adult AML><Adult ANLL><Adult Acute Granulocytic Leukemia><Adult Acute Myeloblastic Leukemia><Adult Acute Myelocytic Leukemia><Adult Acute Myelogenous Leukemia><Adult Acute Myeloid Leukemia><Adult Acute Non-Lymphoblastic Leukemia><Adult Acute Non-Lymphocytic Leukemia><Adult Acute NonLymphoblastic Leukemia><Adult Acute NonLymphocytic Leukemia><Affect><Assay><Auxins><B23><B23 Nuclear Matrix Protein><Base Pairing><Binding><Bioassay><Biological><Biological Assay><Cancer Genes><Cancer-Promoting Gene><Cell Body><Cell Cycle><Cell Cycle Stage><Cell Division Cycle><Cell division><Cells><Cellular Transformation><Chaperone><Chromatin><Chromatin Structure><Compensation><Complex><Cytoplasm><DNA><DNA Methylation><DNA Replication><DNA Synthesis><DNA biosynthesis><DNA methyltransferase 3 alpha mutation><DNA replication fork><DNMT3a><DNMT3a mutation><Deoxyribonucleic Acid><Deposit><Deposition><Disease><Disorder><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Essential Genes><Event><Exhibits><Exons><Expression Signature><Family><Foundations><Funding><Gene Action Regulation><Gene Down-Regulation><Gene Expression><Gene Expression Profile><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Targeting><Genes><Genetic Alteration><Genetic Change><Genetic defect><Grips><HOX gene><Hereditary><Heterochromatin><Histones><Homeo Box Genes><Homeobox Family Gene><Homeobox Genes><Homeodoamin Gene><Homeotic Genes><Human><In Vitro><Individual><Inherited><Investigation><Isoforms><Karyotype><L-Lysine><Label><Lysine><Maintenance><Mammalian Cell><Mediating><Methods><Methylation><Mice><Mice Mammals><Microscopy><Modern Man><Modification><Molecular><Molecular Chaperones><Molecular Interaction><Monitor><Mouse Embryonic Progenitor><Mouse Embryonic Stem Cells><Murine><Mus><Mutate><Mutation><Myeloid Disease><Myeloid Malignancy><Myeloid Neoplasm><Myeloid Tumor><Myeloproliferative Disorders><Myeloproliferative Tumors><Myeloproliferative disease><NPM><NPM1><NPM1 gene><Nucleolar Phosphoprotein B23><Nucleosomes><Oncogenes><Optics><Polycomb><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Protein Isoforms><Protein Modification><Proteins><Proteomics><Reporting><Repression><Role><S Period><S phase><Series><Synthesis Period><Synthesis Phase><Transcript><Transcription Repression><Transcriptional Repression><Transforming Genes><Visualization><Writing><acute granulocytic leukemia><acute granulocytic leukemia cell><acute myeloblastic leukemia cell><acute myelocytic leukemia cell><acute myelogenous leukemia cell><acute myeloid leukemia><acute myeloid leukemia cell><acute nonlymphocytic leukemia cell><biologic><daughter DNA strand><daughter strand><derepression><epigenetic regulation><epigenetically><epigenome><gene expression pattern><gene expression signature><gene repression><genome mutation><grasp><hDNA methyltransferase 3a><in vivo><karyogram><knock-down><knockdown><leukemia><leukemogenesis><metaplastic cell transformation><microscope imaging><microscopic imaging><microscopy imaging><mouse model><murine model><mutant><myeloproliferative neoplasm><nucleolar protein B23><nucleophosmid><nucleophosmin><nucleoplasmin><optical><preservation><protein B23><protein complex><reconstruction><replication fork><segregation><single molecule><social role><synergism><tissue culture><transcriptional profile><transcriptional signature><tumorigenic>