Methylation of the arginine residues of histones by methyltransferases has important
Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however the molecular mechanism(s) of methyltransferase regulation is still unclear as is the biological significance of methylation at particular arginine residues. acid) binds to the substrate (histone) preferentially at the signature motif “KAPRK ” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context inhibition of H3R17 methylation represses expression of or Rabbit Polyclonal to CSFR. is any amino acid (6). CARM1 interacts with GRIP1 (glucocorticoid receptor-interacting protein) and is a secondary coactivator of several nuclear receptors (7 -9). CARM1 also interacts with other chromatin-modifying enzymes such as p300/CREB-binding protein and PRMT1 to bring about cooperative transcriptional activation of p53-responsive genes (10). CARM1 and PRMT1 interactions have also been shown to regulate gene expression in different contexts (11 12 CARM1 is a positive regulator of both (13) and promoter activity (14). CARM1 also participates in various other cellular processes through its ability to methylate nonhistone substrates. Recently CARM1 has been implicated in muscle (15) and T-cell development (6) stem cell differentiation (16) adipocyte differentiation (17) RNA processing (18) and tumorigenesis (19). Despite such broad functional significance the exact molecular mechanisms of the enzyme function are not understood in part due to the unavailability of specific modulators. For example in the case of lysine methyltransferases only two specific inhibitors chaetocin (20) and BIX-01294 (21) are known. However no specific inhibitor for CARM1 with proper characterization is known so far. There is an intensive ongoing effort to identify specific arginine methylation inhibitors (22 -24). Small molecule inhibitors of protein function are powerful tools to probe the physiological roles of enzymes. Furthermore such modulators are potential BMY 7378 lead molecules for therapeutic purposes as evidenced by the recent clinical trials of histone deacetylase inhibitors. Along with the latter the recent discovery of specific and nontoxic small molecule modulators of histone acetyltransferases (HATs) and histone methyltransferases (HMTases) may portend BMY 7378 a new era of epigenetic-based drugs (25). We have established a general screening procedure to identify small molecule modulators of chromatin-modifying enzymes present in plant extracts (obtained from bark stem root or fruit). Using this approach we discovered several small molecule modulators of HATs (25). The same extracts from 25 different plant sources were also screened for HMTase modulatory activity which led to the identification of a molecule (TBBD) having specific activity toward CARM1 as reported here. This small molecule inhibitor TBBD (ellagic acid) shows substrate sequence dependence for enzyme specific inhibition. Furthermore the inhibitor is also active physiologically with a significant consequence on p53-dependent gene expression. MATERIALS AND METHODS Protein Purifications The details of the protein purifications are provided in the supplementary data. Site-directed Mutagenesis Histone H3 point mutants A25P and P16A were obtained by site-directed mutagenesis. The histone H3 expression clone (BL21. Expression and purification of the mutant protein were done as detailed in the supplementary data. HMTase Assay HMTase assay has been performed as described elsewhere (26) also see supplementary data. HAT Assay HAT assays were performed as described elsewhere (26). BMY 7378 Isothermal Titration Calorimetry (ITC) ITC experiments were carried out in a VP-ITC system (Microcal LLC Northampton MA) at 25 °C. Samples were centrifuged and degassed prior to titration. Titration of TBBD against protein (histone H3/CARM1) was carried out by injecting 0.14 mm TBBD in HMTase assay buffer against 0.007 mm histone H3/CARM1. A 2-min interval was allowed between injections for equilibration sufficient for the return of the heat signal BMY 7378 to baseline. A total of 35 injections were carried out to ensure complete titration. The details of the analysis are provided in the supplementary data. Kinetic Characterization of TBBD-mediated Inhibition The HMTase reaction was carried out with CARM1 in the presence of three concentrations of TBBD (10 20 and 50 μm). The HMTase reaction consisted of two substrates histone H3 and the tritiated methyl group donor of AdoMet. In the first assay the concentration of histone H3 was kept.