Quantitative Proteomics Reveals That the Specific Methyltransferases Txr1p and Ezl2p Differentially Affect the Mono-, Di- and Trimethylation States of Histone H3 Lysine 27 (H3K27)

Chunchao Zhang,Anthony J Molascon,Shan Gao,Yifan Liu,Philip C Andrews

doi:10.1074/mcp.m112.021733

Chunchao Zhang, Anthony J Molascon + Show 3 more

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https://doi.org/10.1074/mcp.m112.021733

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Abstract

Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using (15)N metabolically labeled histones as the internal reference. Using this approach, we identified Tetrahymena trithorax related 1 (Txr1p) as a histone methyltransferase in Tetrahymena thermophila and characterized the relationships of the Txr1p and Ezl2p methyltransferases to histone H3 modification. We identified 32 PTMs in more than 60 tryptic peptides from histone H3 of the ciliate model organism Tetrahymena thermophila, and we quantified them (average coefficient of variation: 13%). We examined perturbations to histone modification patterns in two knockout strains of SET-domain-containing histone methyltransferases (HMT). Knockout of TXR1 led to progressively decreased mono-, di-, and tri-methylation of H3K27 and apparent reduced monomethylation of H3K36 in vivo. In contrast, EZL2 knockout resulted in dramatic reductions in both di- and tri-methylation of H3K27 in vivo, whereas the levels of monomethylation of H3K27 increased significantly. This buildup of monomethyl H3K27 is consistent with its role as a substrate for Ezl2p. These results were validated via immunoblotting using modification site-specific antibodies. Taken together, our studies define Txr1p as an H3K27 monomethylation-specific HMT that facilitates the buildup of H3K27 di- and trimethylation by the canonical H3K27-specific HMT, Ezl2p. Our studies also delineate some of the interdependences between various H3 modifications, as compensatory increases in monomethylation at H3K4, H3K23, and H3K56 were also observed for both TXR1 and ELZ2 mutants.

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From the ‡Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109; §Department of Pathology, University of Michigan, Ann Arbor, MI 48109; ʈDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109; **Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109
The functional distinction of the different methylation states is further underscored by the presence of divergent state-specific histone methyltransferases (HMT), such as SETDB1/ SETDB2 and SUV39H1/SUV39H2 [11], SETD8 [12], 1 The abbreviations used are: Ac, acetylation; ATXR, Arabidopsis trithorax related; CV, coefficient of variation; EZL2, enhancer of zestelike 2; HMT, histone methyltransferase; Me1, monomethylation; Me2, dimethylation; Me3, trimethylation; Principal component analysis (PCA), principal component analysis; PHD, plant homeodomain; Pr, propionylation; post-translational modifications (PTMs), post-translational modification; SET, suppressor of variegation, enhancer of zeste, trithorax; TXR1, Tetrahymena trithorax related 1
Along with the immunoblotting validation, constitute a body of evidence strongly supporting the idea that Txr1p is an HMT mainly responsible for H3K27Me1 in Tetrahymena

Summary

Introduction

From the ‡Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109; §Department of Pathology, University of Michigan, Ann Arbor, MI 48109; ʈDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109; **Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109. Arabidopsis trithorax related 5 (ATXR5) and ATXR6, the founding members of a recently identified HMT subfamily, were first isolated as proliferating cell nuclear antigen interacting proteins in Arabidopsis thaliana [17]. Txr1p carries two PHD domains (PHD1 and PHD2), one proliferating cell nuclear antigen interacting protein box (QKLIEDYF), and one C-terminal SET domain (Fig. 1), all of which are consistent with its being a bona fide member of the ATXR5/ATXR6 subfamily of HMTs. In Tetrahymena, there are three homologues of the canonical H3K27-specific HMT enhancer of zeste, referred to as EZL1, EZL2, and EZL3, respectively [24, 25]. EZL2 is expressed at significant levels [24, 25] and required for H3K27 di- and trimethylation in asexually dividing cells (see below)

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