Very little is known about SET-and MYND-containing protein 2 (SMYD2), a member of the SMYD protein family. However, the interest in better understanding the roles of SMYD2 has grown because of recent reports indicating that SMYD2 methylates p53 and histone H3. In this study, we present a combined proteomics and genomics study of SMYD2 designed to elucidate its molecular roles. We report the cytosolic and nuclear interactome of SMYD2 using a combination of immunoprecipitation coupled with high

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... roughput MS, chromatin immunoprecipitation coupled with high throughput MS, and co-immunoprecipitation methods. In particular, we report that SMYD2 interacted with HSP90␣ independently of the SET and MYND domains, with EBP41L3 through the MYND domain, and with p53 through the SET domain. We demonstrated that the interaction of SMYD2 with HSP90␣ enhances SMYD2 histone methyltransferase activity and specificity for histone H3 at lysine 4 (H3K4) in vitro. Interestingly histone H3K36 methyltransferase activity was independent of its interaction with HSP90␣ similar to LSD1 dependence on the androgen receptor. We also showed that the SET domain is required for the methylation at H3K4. We demonstrated using a modified chromatin immunoprecipitation protocol that the SMYD2 gain of function leads to an increase in H3K4 methylation in vivo, whereas no observable levels of H3K36 were detected. We also report that the SMYD2 gain of function was correlated with the upregulation of 37 and down-regulation of four genes, the majority of which are involved in the cell cycle, chromatin remodeling, and transcriptional regulation. TACC2 is one of the genes up-regulated as a result of SMYD2 gain of function. Up-regulation of TACC2 by SMYD2 occurred as a result of SMYD2 binding to the TACC2 promoter where it methylates H3K4. Furthermore the combination of the SMYD2 interactome with the gene expression data suggests that some of the genes regulated by SMYD2 are closely associated with SMYD2-interacting proteins. Molecular & Cellular Proteomics 7:560 -572, 2008. The mapping of protein-protein interaction helps us understand the function of proteins. In recent years, our group (1) and others (2, 3) have performed protein interaction experiments for large sets of human proteins. Our approach for the mapping of protein-protein interactions is based on immunoprecipitation coupled with high throughput MS (IP-HTMS). 1 The SMYD protein family consists of five proteins (SMYD1-5) that are not fully characterized and are grouped based on the presence of two conserved domains (MYND and SET domains). The MYND domain is a zinc finger motif that is involved in protein-protein interaction. It is named after Myeloid, Nervy, and DEAF-1, which are the three most characterized proteins that contain the MYND domain (4). The SET domain is an evolutionarily conserved sequence motif consisting of 130 -140 amino acids. Its name is derived from the three proteins in which it was initially characterized: Su (var) 3-9, Enhancer-of-zeste, and Trithorax (5, 6). These enzymes add methyl groups to lysine residues of histone H3 using S-adenosylmethionine (AdoMet) as a donor substrate (7, 8) . Interest in the SMYD family of proteins has grown significantly because of recent reports indicating that SMYD1, -2, and -3 control gene expression through histone methylation (7-9). In addition, in vitro studies have shown that both SMYD1 and SMYD3 specifically methylate histone at lysine 4 (H3K4) in the presence of HSP90␣ (7, 8) . In contrary to reports on SMYD1 and -3, Brown et al. (9) have shown that SMYD2 dimethylates H3K36 in vitro in the absence of HSP90␣ (9). Beyond their function in histone methylation, proteins containing SET domain were also shown to methylate non-histone proteins. Examples of this activity are the methylation of p53 (10) and the TATA-binding protein-associated factor TAF10 by human SET7/9 (11). Human SET7/9 stabilizes p53 through Lys-372 methylation (10). Similarly SMYD2 regulates p53 activity through methylation at Lys-370, which represses p53-mediated transcriptional regulation (12). These data suggest that p53, like histones, can be activated or repressed through From the