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ã Oncogene (2001) 20, 3014 ± 3020 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Role of protein methylation in chromatin remodeling and transcriptional regulation Michael R Stallcup*,1,2 1 Department of Pathology, University of Southern California, Los Angeles, California, CA 90089, USA; 2Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California, CA 90089, USA Recent ®ndings suggest that lysine and arginine-speci®c methylation of histones may cooperate with other types of post-translational histone modi®cation to regulate chromatin structure and gene transcription. Proteins that methylate histones on arginine residues can collaborate with other coactivators to enhance the activity of speci®c transcriptional activators such as nuclear receptors. Lysine methylation of histones is associated with transcriptionally active nuclei, regulates other types of histone modi®cations, and is necessary for proper mitotic cell divisions. The fact that some transcription factors and proteins involved in RNA processing can also be methylated suggests that protein methylation may also contribute in other ways to regulation of transcription and post-transcriptional steps in gene regulation. In future work, it will be important to develop methods for evaluating the precise roles of protein methylation in the regulation of native genes in physiological settings, e.g. by using chromatin immunoprecipitation assays, dierentiating cell culture systems, and genetically altered cells and animals. It will also be important to isolate additional protein methyltransferases by molecular cloning and to characterize new methyltransferase substrates, the regulation of methyltransferase activities, and the roles of new methyltransferases and substrates. Oncogene (2001) 20, 3014 ± 3020. Keywords: protein methylation; coactivators; histones; transcriptional regulation; chromatin DNA methylation is well known to play roles in the regulation of chromatin structure and regulation of transcription (Bird and Wole, 1999). However, a surprising convergence of recent work in a number of laboratories indicates that protein methylation also contributes to the complex web of mechanisms that govern chromatin remodeling and gene transcription. This review will focus on recent evidence that methylation of histones and perhaps other proteins on lysine and arginine residues cooperates with other types of post-translational modi®cations, such as histone acetylation and phosphorylation, in the regulation of transcription. *Correspondence: MR Stallcup, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los Angeles, California, CA 90089-9092, USA Types of protein methylation and their possible roles in various signaling pathways Protein methylation involves transfer of a methyl group from S-adenosylmethionine to acceptor groups on substrate proteins. Proteins can be methylated on lysine, arginine, histidine, or carboxyl residues (Aletta et al., 1998). In addition, when some aspartate residues in proteins spontaneously convert to isoaspartate as a result of protein aging, a methylation mechanism is used in cells to reverse this process (Najbauer et al., 1996). Speci®c roles for carboxy-methylation of proteins such as Ras and protein phosphatase 2A have been identi®ed in various signaling pathways, and recent evidence has implicated arginine-speci®c protein methyltransferases in several signaling pathways, although in most of these cases the speci®c roles of protein methylation and the relevant protein substrates have not been identi®ed (Aletta et al., 1998; Gary and Clarke, 1998; Lin et al., 1996; Abramovitch et al., 1997). Arginine methylation is important for nuclear export of some hnRNP proteins (Shen et al., 1998; Nichols et al., 2000; Gary and Clarke, 1998). The major arginine methyltransferase in mammalian cells, PRMT1 (Tang et al., 2000a), is required for very early stages of mouse development, suggesting a fundamental role in development (Pawlak et al., 2000). Evidence for dynamic histone methylation The lysine and arginine-rich N-terminal tails of histones are the sites of many types of post-translational modi®cations such as methylation, acetylation, and phosphorylation (Strahl and Allis, 2000; van Holde, 1989). In nucleosomes, these N-terminal histone tails extend beyond the DNA double helix which is wrapped around the nucleosome core formed by the Cterminal regions of eight (two of each type) histone molecules. In their unmodi®ed form the N-terminal histone tails are positively charged and interact with the negatively charged DNA backbone or core histone regions on the same or neighboring nucleosomes; this interaction contributes to chromatin compaction (Luger et al., 1997; Luger and Richmond, 1998). Neutralization of the positive charge of the N-terminal tails by acetylation of lysines and phosphorylation of serines weakens the binding of the N-terminal tail with Protein methylation in transcriptional regulation MR Stallcup the negative regions of nucleosomes and thus contributes to chromatin remodeling. Lysine methylation of histones in vivo is well documented: histone H3 is methylated on lysines 4, 9, 27 and 36, although the speci®c pattern of residues methylated may vary among species; histone H4 is methylated on lysine 20 (van Holde, 1989; Strahl and Allis, 2000). In contrast to lysine methylation, in vivo methylation of arginine residues in histones is less well documented. Arginine methylation has been dicult to detect in native mammalian histones (Gary and Clarke, 1998), but has been reported in Drosophila (Desrosiers and Tanguay, 1988). The latter study found changes in lysine and arginine methylation of histone H3 and in the N-terminal methylation of histone H2B after heat shock of cultured Drosophila cells, suggesting that arginine methylation of histones not only exists but is a dynamically regulated process. Subsequent studies in mammalian and avian cells provided further evidence for association of dynamic histone methylation with active (i.e. acetylated) chromatin, although it was not determined whether the methylation was on lysine or arginine residues (Annunziato et al., 1995; Hendzel and Davie, 1991). The eect of lysine or arginine methylation on chromatin structure is currently unknown. Families of lysine and arginine-speci®c protein methyltransferases Arginine methylation occurs on either or both of the two terminal guanidino nitrogen atoms, resulting in three possible products: monomethylarginine; NG,NGdimethylarginine, in which both methyl groups are on the same nitrogen (asymmetric dimethylarginine); and NG,N'G-dimethylarginine, in which each nitrogen atom receives one methyl group (symmetric dimethylarginine) (Aletta et al., 1998; Gary and Clarke, 1998). cDNA clones for ®ve genetically distinct but related mammalian arginine methyltransferases have been isolated: PRMT1 (Lin et al., 1996), PRMT2/ HRMT1L1 (Scott et al., 1998), PRMT3 (Tang et al., 1998), CARM1 (Chen et al., 1999a), and JBP1 (Pollack et al., 1999). A clone for one yeast protein, Hmt1 or RMT1, belonging to this family has also been identi®ed (Henry and Silver, 1996; Gary et al., 1996). While the overall length of these polypeptide chains varies from 348 ± 608 amino acids, they all share a highly conserved central domain encoding the methyltransferase activity (Zhang et al., 2000). The methylarginine products for PRMT1, PRMT3, RMT1 and CARM1 are monomethylarginine and asymmetric dimethylarginine (Lin et al., 1996; Tang et al., 1998; Gary et al., 1996; BT Schurter et al., 2001, submitted); the type of methylarginine produced by the other two family members has not been reported. Studies conducted in vitro indicated that each of the ®ve mammalian enzymes recognizes a dierent set of protein substrates. PRMT1 prefers to methylate arginine residues in glycine rich regions, which are found in many proteins that bind RNA and are involved in various aspects of RNA metabolism; PRMT1 substrates include ®brillarin, nucleolin, several hnRNPs, and histone H4 (Najbauer et al., 1993; Gary and Clarke, 1998; Chen et al., 1999a). Yeast RMT1 also eciently methylates arginines in glycine rich regions of proteins (Gary and Clarke, 1998). However, CARM1 has little or no activity with the substrates preferred by PRMT1 and RMT1. To date the best substrate reported for CARM1 is histone H3 (Chen et al., 1999a), and the methylation is not in glycine rich regions (BT Schurter et al., 2001, submitted). JBP1 can methylate histones H2A and H4 and myelin basic protein (Pollack et al., 1999). Both PRMT1 and PRMT3 methylated poly(A)-binding protein II, but the two enzymes produced dierent patterns of methylated proteins in a cell extract (Smith et al., 1999; Tang et al., 1998). Substrates for PRMT2 have not yet been reported. Symmetric dimethylarginine has been found in myelin basic protein (Gary and Clarke, 1998) and human spliceosomal Sm proteins D1 and D3, which are components of some of the small nuclear ribonucleoprotein complexes (Brahms et al., 2000), but the enzymes responsible for methylating these proteins have not yet been isolated. Lysines may accept one, two, or three methyl groups on the terminal amine group of the lysine side chain. Histone H3 lysine methyltransferase activity has been observed in several proteins containing SET domains (Rea et al., 2000; O'Carroll et al., 2000). The mammalian SET domain protein SUV39H1 methylates lysine 9 of histone H3, and the SET domain was important for this activity. The ability to methylate histones was observed in some but not all SET domain proteins. It remains to be determined whether the inactive SET domain proteins lack catalytic activity or require undetermined non-histone proteins as substrates, and whether SET domains are common features of lysine methyltransferases. 3015 Recent evidence implicating histone methylation in transcriptional regulation Arginine methylation Recent studies on the mechanism of transcriptional regulation by the nuclear hormone receptors led to the identi®cation of many proteins that may serve as transcriptional coactivators. These coactivators, often working as multi-subunit complexes, bind only to the hormone-activated form of the nuclear receptors and enhance the ability of these hormone-activated transcription factors to activate transcription of target genes (Xu et al., 1999; Westin et al., 2000; McKenna et al., 1999; Freedman, 1999; Glass and Rosenfeld, 2000). Some of the coactivators are involved in chromatin remodeling, by ATP-dependent mechanisms (e.g. SWI/ SNF complex) or histone acetylation (e.g. CBP, p300, p/CAF), while others apparently help to recruit or activate RNA polymerase II and its associated basal transcription factors (e.g. TRAP/DRIP complex). Oncogene Protein methylation in transcriptional regulation MR Stallcup 3016 Oncogene These coactivator complexes constitute signal transduction pathways that transmit the activating signal from the enhancer element-bound nuclear receptors to the transcription machinery, thus enhancing transcription from the associated promoter. One of the best characterized coactivator complexes contains one or more 160 kDa subunits called p160 coactivators; three genetically distinct but related proteins make up the p160 coactivator family (Torchia et al., 1998; Xu et al., 1999). The p160 coactivators have multiple signal input domains that bind directly to hormone activated nuclear receptors and multiple signal output domains which recruit additional coactivators (Ma et al., 1999). The p160 coactivators have an intrinsic protein acetyltransferase activity and recruit additional coactivators such as CBP/p300 and p/CAF, which also possess protein acetyltransferase activities (Chen et al., 1997; Spencer et al., 1997). CBP, p300, and p/CAF can acetylate histones, some DNA-binding transcriptional activator proteins, and some components of the basal transcription machinery (Ogryzko et al., 1996; Gu and Roeder, 1997; Imhof et al., 1997; Korzus et al., 1998; Chen et al., 1999b); p160 proteins can acetylate histones weakly (Chen et al., 1997; Spencer et al., 1997), but eciently acetylated protein substrates for the p160 acetyltransferase activity have not been reported. Histone acetylation plays a major role in nucleosome remodeling (Wole and Guschin, 2000; Cheung et al., 2000b); the physiological role for acetylation of most other protein components of the basal transcription machinery is still under investigation. The search for additional proteins that bind to the p160 coactivators led to the discovery of CARM1, a protein with an arginine-speci®c histone H3 methyltransferase activity which can cooperate with p160 coactivators to enhance the ability of nuclear receptors to activate transcription of transiently transfected reporter genes containing the appropriate nuclear receptor-controlled enhancer elements (Chen et al., 1999a). In vitro CARM1 methylated histone H3 at arginine 2 (minor site) and arginines 17 and 26 (major sites) in the basic N-terminal region, and one or more of four clustered arginines near the C-terminus (BT Schurter et al., 2001, submitted). CARM1 also methylated histone H2A but not the other core histones (Chen et al., 1999a). The N-terminal methylation sites on histone H3 are located among the sites for lysine methylation, lysine acetylation, and serine phosphorylation (Strahl and Allis, 2000). The coactivator and methyltransferase activities of CARM1 led to the proposal that methylation of histones and/or other proteins in the transcription complex cooperates with acetylation of histones and other components of the transcription complex to achieve chromatin remodeling, recruitment of RNA polymerase II, and initiation of transcription. Subsequent transient transfection studies demonstrated a robust synergy between CARM1 and p300 in their functions as coactivators for nuclear receptors (Chen et al., 2000a; Lee et al., 2001). The synergy was only observed with very low levels of transfected nuclear receptor expression vectors and was essentially entirely dependent on the presence of the nuclear receptors, the appropriate hormone, and a p160 coactivator. These studies reinforce the model that p160 coactivators, histone acetyltransferases (e.g. p300 or CBP), and histone methyltransferases function together to make multiple cooperative covalent histone modi®cations that lead to chromatin remodeling. In addition to CARM1, another member of the arginine-speci®c protein methyltransferase family, PRMT1, was also found to function as a coactivator for nuclear receptors in collaboration with p160 coactivators. Furthermore, at low levels of transfected nuclear receptor expression vectors CARM1 and PRMT1 acted synergistically to enhance nuclear receptor function (Koh et al., 2001). Their synergy and activities as coactivators depended upon the cotransfection of a p160 expression vector, consistent with the model that both CARM1 and PRMT1 are recruited to the promoter through contact with the p160 coactivator. Since CARM1 preferentially methylates histone H3 in vitro and PRMT1 methylates histone H4, cooperative methylation of these two histones may be responsible for the observed synergy. Lysine methylation Two recent studies have also implicated lysine methylation of histones in regulation of transcription and chromatin structure. A methyltransferase activity which speci®cally modi®ed lysine 4 of histone H3 in vitro was found in transcriptionally active macronuclei of Tetrahymena but not in transcriptionally inactive micronuclei (Strahl et al., 1999). In the same study methylation of yeast histones was found preferentially on H3 molecules that were also acetylated, thus providing another indication that histone methylation occurs on active chromatin. Results of a separate study suggested a link between methylation of lysine 9 in mammalian histone H3 and the regulation of chromatin structure (Rea et al., 2000). The Su(var) group of genes in Drosophila and S. pombe is a functionally diverse group of genes that were discovered in genetic screens for suppressors of position eect variegation. The Drosophila SU(VAR)3 ± 9 protein is associated with heterochromatin and defects in this protein disrupt the organization of normally heterochromatic regions. Mammalian homologues of this protein (SUV39H) were found to methylate lysine 9 of histone H3. In vitro, methylation of lysine 9 and phosphorylation of serine 10 were mutually antagonistic. Disruption of the two Suv39h genes in mice resulted in decreased viability and developmental abnormalities, and ®broblast cultures from the double null mice had increased levels of serine 10 phosphorylation and aberrant mitotic cell divisions. The above studies suggest that methylation of histones at various sites may play multiple roles in modulating chromatin structure and can be associated with both activation and repression of gene expression. This diversity of Protein methylation in transcriptional regulation MR Stallcup roles for methylation is reminiscent of the ®ndings that histone phosphorylation is apparently involved in regulation of both transcription and mitosis (Strahl and Allis, 2000). Proposed roles for methylation of histones and other proteins in regulation of gene expression; and future directions for investigation Cooperative roles for multiple histone modifications in regulating chromatin structure The speci®c roles for histone methylation, both on lysine and arginine residues, in regulation of chromatin structure and transcription remain to be determined. The accumulated evidence suggests some speci®c models. Acetylation of lysine residues neutralizes the positive charge of the basic N-terminal tails of histones, and thus reduces the binding of the histone tails to DNA or to acidic regions of other proteins. However, since methylation on lysine and arginine residues should not alter their charge, the eect of histone methylation on chromatin structure is dicult to predict. The extra bulk of the methyl group could inhibit protein binding or provide a new epitope for binding of a protein in the same way that acetylation of lysine creates a binding site for proteins with bromodomains (Jacobson et al., 2000). As suggested previously (Strahl and Allis, 2000), the location of speci®c lysine and arginine methylation sites among sites for acetylation and phosphorylation on the N-terminal tails of histones suggests potential cooperation among the dierent types of post-translational histone modi®cations in modulating chromatin structure; speci®c combinations of covalent modi®cations may disrupt and/or promote interactions of the histone tails with DNA and other proteins. The functional synergy between p300 and CARM1 and between PRMT1 and CARM1 as coactivators for nuclear receptors also suggests a possible cooperation between acetylation and methylation of various histones (Chen et al., 2000a; Koh et al., 2001; Lee et al., 2001). Methylation could also in¯uence the eciency with which other types of covalent histone modi®cations occur, as illustrated already by the negative eect that methylation of lysine 9 of histone H3 has on phosphorylation of serine 10 (Rea et al., 2000) and the cooperative eect on transcriptional activation by acetylation and phosphorylation of lysine 14 and serine 10, respectively, on histone H3 (Cheung et al., 2000a; Lo et al., 2000). Thus far, the activities of most methyltransferases have been examined only with free histone substrates. Intact nucleosomes must also be tested as substrates. Role of protein methylation in the coactivator function of protein methyltransferases The mechanism for recruitment of lysine methyltransferases to the promoter of activated genes must await the cloning of cDNAs encoding these methyltransferases. Since there is solid evidence for methyllysine in histones and exciting recent ®ndings which suggest a role for lysine-speci®c methylation in regulation of chromatin structure and transcription, identi®cation of the responsible enzymes must obviously be an important goal for future research. The activities of these enzymes on histones will almost certainly be regulated as part of signaling pathways that regulate chromatin structure and transcription. Identi®cation of the methyltransferases will provide an entry point for studying the signaling pathways and how they ®t into the increasingly complex picture of chromatin structure and transcriptional regulation. Possible mechanisms for preferential arginine methylation of active chromatin are suggested by the physical and functional relationships among the various nuclear receptor coactivators in transient transfection experiments. Methyltransferases CARM1 and PRMT1 bind to a C-terminal activation domain (AD2) of p160 coactivators and can only function as nuclear receptor coactivators when co-expressed with a p160 coactivator with an intact AD2 region (Chen et al., 1999a, 2000a). The function of p300 as a nuclear receptor coactivator is similarly dependent on co-expression of a p160 coactivator with an intact AD1 region, which is the binding site for p300 (Chen et al., 2000a; Li et al., 2000). These results suggest that both methyltransferases and acetyltransferases are recruited to the promoter through their physical interaction with speci®c domains of p160 coactivators, which are recruited to the promoter by direct interactions with the DNA-bound nuclear receptors. Thus far, the activities of CARM1 and PRMT1 as coactivators for nuclear receptors have only been demonstrated with transiently transfected reporter genes. It will be important to test whether these proteins are required for or can enhance nuclear receptor function on target genes in more physiologically relevant settings. Transient transfection experiments in cells containing stably integrated reporter genes oer one such setting. Chromatin immunoprecipitation, using antibodies against methyltransferase proteins, can be used to test whether these proteins are recruited to the promoter of native target genes when they are activated by nuclear receptors and their hormones. Such assays have already been used to show recruitment of other types of nuclear receptor coactivators to target genes (Chen et al., 1999b; Shang et al., 2000). Similarly, it will be important to test directly whether methylated histones are associated with speci®c genes in the active versus the quiescent state. Strategies that were used to show de®nitively the involvement of histone acetylation in transcriptional regulation should be applicable to the study of histone methylation. Antibodies that recognize acetylated but not unmodi®ed histones have been used in chromatin immunoprecipitation assays to demonstrate that histone acetylation occurs in conjunction with transcriptional activation of speci®c promoters (Chen et al., 1999b; Cheung et al., 2000a). Since some speci®c in 3017 Oncogene Protein methylation in transcriptional regulation MR Stallcup 3018 Oncogene vitro sites of lysine and arginine methylation on histones have been determined already, similar strategies should be applied to the study of histone methylation. In conjunction with these studies, it will be important to use recently improved mass spectrometry technology to look for more concrete evidence to support the existence of arginine methylation of histones in vivo, and to test whether the sites of lysine and arginine methylation in vivo correlate with those observed with speci®c methyltransferases in vitro. Various procedures to accomplish a functional knock-out of the methyltransferase proteins or their enzymatic activities should prove to be useful for assessing their biological functions. Of course, the yeast system is the most convenient for this type of experiment, and such experiments have already shown that Hmt1/RMT1 and its methyltransferase activity are important in RNA metabolism and for the nuclearcytoplasmic shuttling of speci®c RNA binding proteins (Shen et al., 1998; McBride et al., 2000). However, the larger number of arginine-speci®c methyltransferase proteins in mammalian cells than in yeast indicates that there may be more complex roles for these proteins in the higher eukaryotes. In mammalian cells antisense technology and dominant negative mutants of CARM1 and PRMT1 (if they can be developed) should prove helpful in testing whether these proteins are required for transcriptional activation by nuclear receptors. Studies in dierentiating cell culture models, such as one which demonstrated a role for p160 coactivator GRIP1 in a developing myocyte system (Chen et al., 2000b), may also prove useful. Genetic knockout and over-expression in transgenic mice will also help to determine the roles of these proteins in hormone action and speci®c physiological processes; studies already performed with the p160 coactivators can serve as a model for such studies (Xu et al., 1998, 2000). A mouse lacking functional PRMT1 has already been established, and the phenotype of the homozygous null mouse is embryonic lethal (Pawlak et al., 2000); but the eect of this mutation on nuclear receptor function and chromatin structure has not been examined. The fact that CARM1 and PRMT1 have argininespeci®c protein methyltransferase activities suggests that protein methylation may be involved in their coactivator activities. In an initial genetic test of this hypothesis, substitution of alanine for three highly conserved residues (VLD) in the region of CARM1 responsible for S-adenosylmethionine binding resulted in loss of methyltransferase and coactivator activities, thus supporting a role for protein methylation in the coactivator function of CARM1 (Chen et al., 1999a). However, a recent X-ray crystal structure of the catalytic domain of PRMT3, which is closely related in sequence to that of CARM1, indicates that the VLD residues are probably involved in intramolecular folding and thus may aect more than just Sadenosylmethionine binding (Zhang et al., 2000). Ideal mutations for testing the role of methylation in the coactivator function of CARM1 should selectively eliminate binding of S-adenosylmethionine, protein substrate, or p160 coactivator by CARM1 without aecting overall three-dimensional structure. The X-ray crystal structure for PRMT3 and another of yeast Hmt1/RMT1 (Weiss et al., 2000) provide new insights into the binding of S-adenosylmethionine and the target arginine residue of the protein substrate and thus should help to guide design of more suitable mutations for testing the role of methyltransferase activity in coactivator function. Methylation of non-histone substrates The possibility that the lysine and arginine-speci®c methyltransferases contribute to gene activation by methylation of protein substrates in addition to histones must also be considered. The histone acetyltransferases p300, CBP, and p/CAF all have the ability to acetylate speci®c non-histone proteins found in the transcription machinery, such as other coactivators, transcriptional activators, and basal transcription factors (Ogryzko et al., 1996; Gu and Roeder, 1997; Imhof et al., 1997; Korzus et al., 1998; Chen et al., 1999b). By analogy, methyltransferases recruited to active promoters may have non-histone targets as well. In fact, PRMT1 and the yeast Hmt1/RMT1 can methylate many RNA binding proteins which are known to be involved in various aspects of RNA processing, transport, translation, and metabolism (Najbauer et al., 1993; Lin et al., 1996; Gary and Clarke, 1998; Aletta et al., 1998; Shen et al., 1998; Smith et al., 1999; Tang et al., 2000b; Nichols et al., 2000). The fact that transcriptional initiation and many post-transcriptional steps are known to be coupled and coordinated (Monsalve et al., 2000) suggests that some proteins currently classi®ed as transcriptional coactivators could actually function by facilitating post-transcriptional steps. For example, recruitment of PRMT1 to the promoter through its contact with p160 coactivators could enhance reporter gene expression by methylation of (e.g.) histone H4, an RNA processing factor that is associated with the transcription initiation complex, or both. In that regard, it is interesting that another coactivator, PGC-1 contains RNA binding motifs and has been shown to help stimulate and couple transcription initiation and subsequent splicing of the transcript (Monsalve et al., 2000). In addition the yeast Hmt1/RMT1 has been shown to interact genetically with proteins involved in RNA processing and transport (Henry and Silver, 1996; Shen et al., 1998), and the methyltransferase activity of Hmt1/ RMT1 is important for this function in yeast (McBride et al., 2000). A number of unidenti®ed proteins in cell extracts have been shown to be substrates for methylation by speci®c or unspeci®ed methyltransferases (Lin et al., 1996; Tang et al., 1998; Frankel and Clarke, 1999; Pollack et al., 1999). Thus, it is likely that additional methylation targets involved in regulation of transcription or processes coupled to transcription remain to be identi®ed. As new methyltransferase substrates are identi®ed, it will be desirable to address the physiological roles of their methylation. In order to do this, it will be Protein methylation in transcriptional regulation MR Stallcup necessary to know speci®c functions for the substrate proteins and to have speci®c assays to detect these functions (e.g. cell free functional assays, transfection assays in cultured cells, or gene replacement strategies in living organisms). In that case, the importance of methylation could be addressed by ®rst determining the methylation site(s) and modifying them genetically (e.g. change lysine to arginine or arginine to lysine). The activities of the mutants which cannot be methylated could then be compared with the wild type protein in the available assay. Reversibility of protein methylation Most steps in signal transduction pathways are readily reversible so that the biological response to the stimulus can be shut o after removal of the stimulus. Thus, if protein methylation is involved in signal transduction, it seems necessary to assume that the methylation is reversible or that the methylated protein can be eliminated or replaced by an unmethylated protein. However, little is known about this process. The ®ndings that at least some methylation of histones is dynamic rather than static implies that methylation must be reversible. Methylated histones can thus be used as substrates to search for enzymes that remove the methyl groups. An activity that removes epsilon-methyl groups from methyllysine of histones or from free methyllysine has been reported (Paik and Kim, 1973, 1974), but there is no evidence for an activity that can remove methyl groups from arginine residues of proteins. For other types of covalent modi®cation, such as acetylation and phosphorylation, both the addition and the removal of the modifying moiety are highly regulated. 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