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Studies of the Epigenetic Regulator NSD2 by Targeted and Quantitative Mass Spectrometry

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Epigenetics is the study of chromatin-based events that regulate gene expression without the change of DNA sequence, including DNA methylation, histone modification and chromatin remodeling. Epigenetic regulators are encoded to modify chromatin in a highly regulated and dynamic manner. A growing number of studies have suggested the dysregulation of epigenetic regulators through chromosomal translocations, mutations, and deletions is highly associated with tumorigenesis. NSD2 (MMSET/WHSC1) is a histone methyltransferase that catalyzes di-methylation of histone H3 lysine 36 (H3K36me2), a mark associated with gene activation. The overexpression of NSD2 in t(4;14)+ multiple myeloma (MM) leads to aberrant gene expression and poor prognosis. We identified a recurrent gain-of-function mutation E1099K of NSD2 in pediatric acute lymphoblastic leukemia (ALL) that alters global histone modification landscape and gene expression pattern. Taking advantage of the novel gene-editing CRISPR/Cas9 system and SILAC-based quantitative mass spectrometry, we studied the kinetics of histone H3K36me2 in wildtype and E1099K mutant ALL cell lines. The mutant NSD2 enzyme showed increased activity catalyzing monomethylation of histone H3K36, which is the rate-limiting step. Combining histone methyltransferase assay and quantitative mass spectrometry, we found that the E1099K mutant NSD2 enzyme has elevated turnover rate at the monomethylation step in vitro. To better understand the molecular functions of NSD2, we adopted BioID proximity-based biotinylation technique, coupled with label-free quantitative mass spectrometry to map NSD2 interaction network. The BioID labeling strategy helped us to capture transient and weak interactions that are otherwise lost during affinity purification. Meanwhile, the label-free quantitative mass spectrometry empowered us to filter out noises from random tagging. Twenty-four core NSD2 nuclear interacting partners were discovered in this study with high confidence. They are involved in multiple molecular pathways and biological process including chromatin remodeling, gene expression regulation, DNA damage repair, and RNA splicing. One of the targets, PARP1, a molecular sensor in respond to DNA breaks, was further validated by co-immunoprecipitation. PARP1 and NSD2 have been previously found to be recruited to DNA double strand breaks (DSBs) upon damage and H3K36me2 marks are enriched at damage sites. We demonstrated that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggested the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, we showed that PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2 regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. Overall, newly available techniques were utilized in this study to understand the enzymatic activity and biological functions of NSD2 in hematological malignances. This work provides evidence of crosstalk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation and other pathways.

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