Dataset Information

Function of the MYND domain and C-terminal region in regulating the subcellular localization and catalytic activity of the SMYD family lysine methyltransferase Set5

ABSTRACT: SMYD lysine methyltransferases target histones and non-histone proteins for methylation and are critical regulators of muscle developmental and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. Saccharomyces cerevisiae contains two SMYD family members, Set5 and Set6, which share similar structural elements with the mammalian SMYD proteins. Set5 is a histone H4 lysine 5, 8, and 12 methyltransferase, implicated in the regulation of stress responses and genome stability. While the SMYD proteins have diverse roles in cells, there are many gaps in our understanding of how these enzymes are regulated. Here, we performed mutational analysis of Set5, combined with phosphoproteomics, to identify regulatory mechanisms for its enzymatic activity and subcellular localization. Our results indicate that the MYND domain promotes Set5 chromatin association in cells and mediates its interaction with DNA. Phosphoproteomics revealed extensive phosphorylation of Set5 and phosphomimetic mutations enhance Set5 catalytic activity and diminish its ability to interact with chromatin in cells. These studies uncover multiple regions within Set5 that regulate its localization and methyltransferase activity and highlight potential avenues for understanding mechanisms controlling the diverse roles of SMYD enzymes.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Whole Body

SUBMITTER: James Moresco

LAB HEAD: Erin. M. Green

PROVIDER: PXD014756 | Pride | 2019-11-11

REPOSITORIES: Pride

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Publications

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.

Jaiswal Deepika D Turniansky Rashi R Moresco James J JJ Ikram Sabeen S Ramaprasad Ganesh G Akinwole Assefa A Wolf Julie J Yates John R JR Green Erin M EM

Molecular and cellular biology 20200103 2

SMYD lysine methyltransferases target histones and nonhistone proteins for methylation and are critical regulators of muscle development and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. <i>Saccharomyces cerevisiae</i> contains two SMYD proteins, Set5 and Set6, which share structural elements with the mammalian SMYD enzymes. Set5 is a histone H4 lysine 5, 8, and ...[more]

PMID: 31685550

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Project description:We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma. 10 DLBCL cell lines (7 mutant and 3 wild type EZH2), that were differentially sensitive to GSK126 in proliferation assays, were treated for 72 hours, in duplicate (n=2), with either DMSO (vehicle) or 500nM of GSK126, a potent selective EZH2 inhibitor. EZH2 mutant cell lines are Pfeiffer, KARPAS-422, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB and SU-DHL-4. EZH2 wildtype cell lines are HT, OCI-LY-19 and Toledo.

Dataset Information

Function of the MYND domain and C-terminal region in regulating the subcellular localization and catalytic activity of the SMYD family lysine methyltransferase Set5

Publications

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.

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