Role of Protein Methylation in Chromatin Remodeling and Transcriptional Regulation

Role of protein methylation in chromatin remodeling and transcriptional regulation

Michael R Stallcup*,1,2

1Department 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 Types of protein methylation and their possible roles in methylation of histones may cooperate with other types of various signaling pathways post-translational histone modi®cation to regulate chromatin structure and gene transcription. Proteins that Protein methylation involves transfer of a methyl methylate histones on arginine residues can collaborate group from S-adenosylmethionine to acceptor groups with other coactivators to enhance the activity of speci®c on substrate proteins. Proteins can be methylated on transcriptional activators such as nuclear receptors. Lysine lysine, arginine, histidine, or carboxyl residues (Aletta methylation of histones is associated with transcriptionally et al., 1998). In addition, when some aspartate residues active nuclei, regulates other types of histone modi®ca- in proteins spontaneously convert to isoaspartate as a tions, and is necessary for proper mitotic cell divisions. The result of protein aging, a methylation mechanism is fact that some transcription factors and proteins involved used in cells to reverse this process (Najbauer et al., in RNA processing can also be methylated suggests that 1996). Speci®c roles for carboxy-methylation of protein methylation may also contribute in other ways to proteins such as Ras and protein phosphatase 2A have regulation of transcription and post-transcriptional steps been identi®ed in various signaling pathways, and in gene regulation. In future work, it will be important to recent evidence has implicated arginine-speci®c protein develop methods for evaluating the precise roles of protein methyltransferases in several signaling pathways, methylation in the regulation of native genes in physiolo- although in most of these cases the speci®c roles of gical settings, e.g. by using chromatin immunoprecipita- protein methylation and the relevant protein substrates tion assays, dierentiating cell culture systems, and have not been identi®ed (Aletta et al., 1998; Gary and genetically altered cells and animals. It will also be Clarke, 1998; Lin et al., 1996; Abramovitch et al., important to isolate additional protein methyltransferases 1997). Arginine methylation is important for nuclear by molecular cloning and to characterize new methyl- export of some hnRNP proteins (Shen et al., 1998; transferase substrates, the regulation of methyltransferase Nichols et al., 2000; Gary and Clarke, 1998). The activities, and the roles of new methyltransferases and major arginine methyltransferase in mammalian cells, substrates. Oncogene (2001) 20, 3014 ± 3020. PRMT1 (Tang et al., 2000a), is required for very early stages of mouse development, suggesting a funda- Keywords: protein methylation; coactivators; histones; mental role in development (Pawlak et al., 2000). transcriptional regulation; chromatin

Evidence for dynamic histone methylation DNA methylation is well known to play roles in the regulation of chromatin structure and regulation of The lysine and arginine-rich N-terminal tails of transcription (Bird and Wole, 1999). However, a histones are the sites of many types of post-transla- surprising convergence of recent work in a number of tional modi®cations such as methylation, acetylation, laboratories indicates that protein methylation also and phosphorylation (Strahl and Allis, 2000; van contributes to the complex web of mechanisms that Holde, 1989). In nucleosomes, these N-terminal histone govern chromatin remodeling and gene transcription. tails extend beyond the DNA double helix which is This review will focus on recent evidence that wrapped around the nucleosome core formed by the C- methylation of histones and perhaps other proteins terminal regions of eight (two of each type) histone on lysine and arginine residues cooperates with other molecules. In their unmodi®ed form the N-terminal types of post-translational modi®cations, such as histone tails are positively charged and interact with histone acetylation and phosphorylation, in the regula- the negatively charged DNA backbone or core histone tion of transcription. 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 *Correspondence: MR Stallcup, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los tails by acetylation of lysines and phosphorylation of Angeles, California, CA 90089-9092, USA serines weakens the binding of the N-terminal tail with Protein methylation in transcriptional regulation MR Stallcup 3015 the negative regions of nucleosomes and thus con- metabolism; PRMT1 substrates include ®brillarin, tributes to chromatin remodeling. nucleolin, several hnRNPs, and histone H4 (Najbauer Lysine methylation of histones in vivo is well et al., 1993; Gary and Clarke, 1998; Chen et al., 1999a). documented: histone H3 is methylated on lysines 4, 9, Yeast RMT1 also eciently methylates arginines in 27 and 36, although the speci®c pattern of residues glycine rich regions of proteins (Gary and Clarke, methylated may vary among species; histone H4 is 1998). However, CARM1 has little or no activity with methylated on lysine 20 (van Holde, 1989; Strahl and the substrates preferred by PRMT1 and RMT1. To Allis, 2000). In contrast to lysine methylation, in vivo date the best substrate reported for CARM1 is histone methylation of arginine residues in histones is less well H3 (Chen et al., 1999a), and the methylation is not in documented. Arginine methylation has been dicult to glycine rich regions (BT Schurter et al., 2001, detect in native mammalian histones (Gary and Clarke, submitted). JBP1 can methylate histones H2A and H4 1998), but has been reported in Drosophila (Desrosiers and myelin basic protein (Pollack et al., 1999). Both and Tanguay, 1988). The latter study found changes in PRMT1 and PRMT3 methylated poly(A)-binding lysine and arginine methylation of histone H3 and in protein II, but the two enzymes produced dierent the N-terminal methylation of histone H2B after heat patterns of methylated proteins in a cell extract (Smith shock of cultured Drosophila cells, suggesting that et al., 1999; Tang et al., 1998). Substrates for PRMT2 arginine methylation of histones not only exists but is a have not yet been reported. dynamically regulated process. Subsequent studies in Symmetric dimethylarginine has been found in mammalian and avian cells provided further evidence myelin basic protein (Gary and Clarke, 1998) and for association of dynamic histone methylation with human spliceosomal Sm proteins D1 and D3, which active (i.e. acetylated) chromatin, although it was not are components of some of the small nuclear determined whether the methylation was on lysine or ribonucleoprotein complexes (Brahms et al., 2000), arginine residues (Annunziato et al., 1995; Hendzel and but the enzymes responsible for methylating these Davie, 1991). The eect of lysine or arginine methyla- proteins have not yet been isolated. tion on chromatin structure is currently unknown. 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 Families of lysine and arginine-speci®c protein observed in several proteins containing SET domains methyltransferases (Rea et al., 2000; O'Carroll et al., 2000). The mammalian SET domain protein SUV39H1 methylates Arginine methylation occurs on either or both of the lysine 9 of histone H3, and the SET domain was two terminal guanidino nitrogen atoms, resulting in important for this activity. The ability to methylate three possible products: monomethylarginine; NG,NG- histones was observed in some but not all SET domain dimethylarginine, in which both methyl groups are on proteins. It remains to be determined whether the the same nitrogen (asymmetric dimethylarginine); and inactive SET domain proteins lack catalytic activity or NG,N'G-dimethylarginine, in which each nitrogen atom require undetermined non-histone proteins as sub- receives one methyl group (symmetric dimethylargi- strates, and whether SET domains are common nine) (Aletta et al., 1998; Gary and Clarke, 1998). features of lysine methyltransferases. cDNA clones for ®ve genetically distinct but related mammalian arginine methyltransferases have been isolated: PRMT1 (Lin et al., 1996), PRMT2/ Recent evidence implicating histone methylation in HRMT1L1 (Scott et al., 1998), PRMT3 (Tang et al., transcriptional regulation 1998), CARM1 (Chen et al., 1999a), and JBP1 (Pollack et al., 1999). A clone for one yeast protein, Hmt1 or Arginine methylation RMT1, belonging to this family has also been identi®ed (Henry and Silver, 1996; Gary et al., 1996). While the Recent studies on the mechanism of transcriptional overall length of these polypeptide chains varies from regulation by the nuclear hormone receptors led to the 348 ± 608 amino acids, they all share a highly conserved identi®cation of many proteins that may serve as central domain encoding the methyltransferase activity transcriptional coactivators. These coactivators, often (Zhang et al., 2000). The methylarginine products for working as multi-subunit complexes, bind only to the PRMT1, PRMT3, RMT1 and CARM1 are mono- hormone-activated form of the nuclear receptors and methylarginine and asymmetric dimethylarginine (Lin enhance the ability of these hormone-activated tran- et al., 1996; Tang et al., 1998; Gary et al., 1996; BT scription factors to activate transcription of target Schurter et al., 2001, submitted); the type of methyl- genes (Xu et al., 1999; Westin et al., 2000; McKenna et arginine produced by the other two family members al., 1999; Freedman, 1999; Glass and Rosenfeld, 2000). has not been reported. Studies conducted in vitro Some of the coactivators are involved in chromatin indicated that each of the ®ve mammalian enzymes remodeling, by ATP-dependent mechanisms (e.g. SWI/ recognizes a dierent set of protein substrates. PRMT1 SNF complex) or histone acetylation (e.g. CBP, p300, prefers to methylate arginine residues in glycine rich p/CAF), while others apparently help to recruit or regions, which are found in many proteins that bind activate RNA polymerase II and its associated basal RNA and are involved in various aspects of RNA transcription factors (e.g. TRAP/DRIP complex).

Oncogene Protein methylation in transcriptional regulation MR Stallcup 3016 These coactivator complexes constitute signal transduc- only observed with very low levels of transfected tion pathways that transmit the activating signal from nuclear receptor expression vectors and was essentially the enhancer element-bound nuclear receptors to the entirely dependent on the presence of the nuclear transcription machinery, thus enhancing transcription receptors, the appropriate hormone, and a p160 from the associated promoter. coactivator. These studies reinforce the model that One of the best characterized coactivator complexes p160 coactivators, histone acetyltransferases (e.g. p300 contains one or more 160 kDa subunits called p160 or CBP), and histone methyltransferases function coactivators; three genetically distinct but related together to make multiple cooperative covalent histone proteins make up the p160 coactivator family (Torchia modi®cations that lead to chromatin remodeling. et al., 1998; Xu et al., 1999). The p160 coactivators In addition to CARM1, another member of the have multiple signal input domains that bind directly arginine-speci®c protein methyltransferase family, to hormone activated nuclear receptors and multiple PRMT1, was also found to function as a coactivator signal output domains which recruit additional coacti- for nuclear receptors in collaboration with p160 vators (Ma et al., 1999). The p160 coactivators have an coactivators. Furthermore, at low levels of transfected intrinsic protein acetyltransferase activity and recruit nuclear receptor expression vectors CARM1 and additional coactivators such as CBP/p300 and p/CAF, PRMT1 acted synergistically to enhance nuclear which also possess protein acetyltransferase activities receptor function (Koh et al., 2001). Their synergy (Chen et al., 1997; Spencer et al., 1997). CBP, p300, and activities as coactivators depended upon the co- and p/CAF can acetylate histones, some DNA-binding transfection of a p160 expression vector, consistent transcriptional activator proteins, and some compo- with the model that both CARM1 and PRMT1 are nents of the basal transcription machinery (Ogryzko et recruited to the promoter through contact with the al., 1996; Gu and Roeder, 1997; Imhof et al., 1997; p160 coactivator. Since CARM1 preferentially methy- Korzus et al., 1998; Chen et al., 1999b); p160 proteins lates histone H3 in vitro and PRMT1 methylates can acetylate histones weakly (Chen et al., 1997; histone H4, cooperative methylation of these two Spencer et al., 1997), but eciently acetylated protein histones may be responsible for the observed synergy. substrates for the p160 acetyltransferase activity have not been reported. Histone acetylation plays a major Lysine methylation role in nucleosome remodeling (Wole and Guschin, 2000; Cheung et al., 2000b); the physiological role for Two recent studies have also implicated lysine acetylation of most other protein components of the methylation of histones in regulation of transcription basal transcription machinery is still under investiga- and chromatin structure. A methyltransferase activity tion. which speci®cally modi®ed lysine 4 of histone H3 in The search for additional proteins that bind to the vitro was found in transcriptionally active macronuclei p160 coactivators led to the discovery of CARM1, a of Tetrahymena but not in transcriptionally inactive protein with an arginine-speci®c histone H3 methyl- micronuclei (Strahl et al., 1999). In the same study transferase activity which can cooperate with p160 methylation of yeast histones was found preferentially coactivators to enhance the ability of nuclear receptors on H3 molecules that were also acetylated, thus to activate transcription of transiently transfected providing another indication that histone methylation reporter genes containing the appropriate nuclear occurs on active chromatin. Results of a separate study receptor-controlled enhancer elements (Chen et al., suggested a link between methylation of lysine 9 in 1999a). In vitro CARM1 methylated histone H3 at mammalian histone H3 and the regulation of chroma- arginine 2 (minor site) and arginines 17 and 26 (major tin structure (Rea et al., 2000). The Su(var) group of sites) in the basic N-terminal region, and one or more genes in Drosophila and S. pombe is a functionally of four clustered arginines near the C-terminus (BT diverse group of genes that were discovered in genetic Schurter et al., 2001, submitted). CARM1 also screens for suppressors of position eect variegation. methylated histone H2A but not the other core The Drosophila SU(VAR)3 ± 9 protein is associated histones (Chen et al., 1999a). The N-terminal methyla- with heterochromatin and defects in this protein tion sites on histone H3 are located among the sites for disrupt the organization of normally heterochromatic lysine methylation, lysine acetylation, and serine regions. Mammalian homologues of this protein phosphorylation (Strahl and Allis, 2000). The coacti- (SUV39H) were found to methylate lysine 9 of histone vator and methyltransferase activities of CARM1 led H3. In vitro, methylation of lysine 9 and phosphoryla- to the proposal that methylation of histones and/or tion of serine 10 were mutually antagonistic. Disrup- other proteins in the transcription complex cooperates tion of the two Suv39h genes in mice resulted in with acetylation of histones and other components of decreased viability and developmental abnormalities, the transcription complex to achieve chromatin and ®broblast cultures from the double null mice had remodeling, recruitment of RNA polymerase II, and increased levels of serine 10 phosphorylation and initiation of transcription. aberrant mitotic cell divisions. The above studies Subsequent transient transfection studies demon- suggest that methylation of histones at various sites strated a robust synergy between CARM1 and p300 may play multiple roles in modulating chromatin in their functions as coactivators for nuclear receptors structure and can be associated with both activation (Chen et al., 2000a; Lee et al., 2001). The synergy was and repression of gene expression. This diversity of

Oncogene Protein methylation in transcriptional regulation MR Stallcup 3017 roles for methylation is reminiscent of the ®ndings that the cloning of cDNAs encoding these methyltrans- histone phosphorylation is apparently involved in ferases. Since there is solid evidence for methyllysine in regulation of both transcription and mitosis (Strahl histones and exciting recent ®ndings which suggest a and Allis, 2000). 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 Proposed roles for methylation of histones and other enzymes on histones will almost certainly be regulated proteins in regulation of gene expression; and future as part of signaling pathways that regulate chromatin directions for investigation structure and transcription. Identi®cation of the methyltransferases will provide an entry point for Cooperative roles for multiple histone modifications in studying the signaling pathways and how they ®t into regulating chromatin structure the increasingly complex picture of chromatin structure The speci®c roles for histone methylation, both on and transcriptional regulation. lysine and arginine residues, in regulation of chromatin Possible mechanisms for preferential arginine methy- structure and transcription remain to be determined. lation of active chromatin are suggested by the physical The accumulated evidence suggests some speci®c and functional relationships among the various nuclear models. Acetylation of lysine residues neutralizes the receptor coactivators in transient transfection experi- positive charge of the basic N-terminal tails of ments. Methyltransferases CARM1 and PRMT1 bind histones, and thus reduces the binding of the histone to a C-terminal activation domain (AD2) of p160 tails to DNA or to acidic regions of other proteins. coactivators and can only function as nuclear receptor However, since methylation on lysine and arginine coactivators when co-expressed with a p160 coactivator residues should not alter their charge, the eect of with an intact AD2 region (Chen et al., 1999a, 2000a). histone methylation on chromatin structure is dicult The function of p300 as a nuclear receptor coactivator to predict. The extra bulk of the methyl group could is similarly dependent on co-expression of a p160 inhibit protein binding or provide a new epitope for coactivator with an intact AD1 region, which is the binding of a protein in the same way that acetylation binding site for p300 (Chen et al., 2000a; Li et al., of lysine creates a binding site for proteins with 2000). These results suggest that both methyltrans- bromodomains (Jacobson et al., 2000). ferases and acetyltransferases are recruited to the As suggested previously (Strahl and Allis, 2000), the promoter through their physical interaction with location of speci®c lysine and arginine methylation sites speci®c domains of p160 coactivators, which are among sites for acetylation and phosphorylation on the recruited to the promoter by direct interactions with N-terminal tails of histones suggests potential coopera- the DNA-bound nuclear receptors. tion among the dierent types of post-translational Thus far, the activities of CARM1 and PRMT1 as histone modi®cations in modulating chromatin struc- coactivators for nuclear receptors have only been ture; speci®c combinations of covalent modi®cations demonstrated with transiently transfected reporter may disrupt and/or promote interactions of the histone genes. It will be important to test whether these tails with DNA and other proteins. The functional proteins are required for or can enhance nuclear synergy between p300 and CARM1 and between receptor function on target genes in more physiologi- PRMT1 and CARM1 as coactivators for nuclear cally relevant settings. Transient transfection experi- receptors also suggests a possible cooperation between ments in cells containing stably integrated reporter acetylation and methylation of various histones (Chen genes oer one such setting. Chromatin immunopreci- et al., 2000a; Koh et al., 2001; Lee et al., 2001). pitation, using antibodies against methyltransferase Methylation could also in¯uence the eciency with proteins, can be used to test whether these proteins which other types of covalent histone modi®cations are recruited to the promoter of native target genes occur, as illustrated already by the negative eect that when they are activated by nuclear receptors and their methylation of lysine 9 of histone H3 has on hormones. Such assays have already been used to show phosphorylation of serine 10 (Rea et al., 2000) and recruitment of other types of nuclear receptor the cooperative eect on transcriptional activation by coactivators to target genes (Chen et al., 1999b; Shang acetylation and phosphorylation of lysine 14 and serine et al., 2000). Similarly, it will be important to test 10, respectively, on histone H3 (Cheung et al., 2000a; directly whether methylated histones are associated Lo et al., 2000). Thus far, the activities of most with speci®c genes in the active versus the quiescent methyltransferases have been examined only with free state. Strategies that were used to show de®nitively the histone substrates. Intact nucleosomes must also be involvement of histone acetylation in transcriptional tested as substrates. regulation should be applicable to the study of histone methylation. Antibodies that recognize acetylated but not unmodi®ed histones have been used in chromatin Role of protein methylation in the coactivator function of immunoprecipitation assays to demonstrate that his- protein methyltransferases tone acetylation occurs in conjunction with transcrip- The mechanism for recruitment of lysine methyltrans- tional activation of speci®c promoters (Chen et al., ferases to the promoter of activated genes must await 1999b; Cheung et al., 2000a). Since some speci®c in

Oncogene Protein methylation in transcriptional regulation MR Stallcup 3018 vitro sites of lysine and arginine methylation on substrate, or p160 coactivator by CARM1 without histones have been determined already, similar strate- aecting overall three-dimensional structure. The X-ray gies should be applied to the study of histone crystal structure for PRMT3 and another of yeast methylation. In conjunction with these studies, it will Hmt1/RMT1 (Weiss et al., 2000) provide new insights be important to use recently improved mass spectro- into the binding of S-adenosylmethionine and the metry technology to look for more concrete evidence to target arginine residue of the protein substrate and support the existence of arginine methylation of thus should help to guide design of more suitable histones in vivo, and to test whether the sites of lysine mutations for testing the role of methyltransferase and arginine methylation in vivo correlate with those activity in coactivator function. observed with speci®c methyltransferases in vitro. Various procedures to accomplish a functional Methylation of non-histone substrates knock-out of the methyltransferase proteins or their enzymatic activities should prove to be useful for The possibility that the lysine and arginine-speci®c assessing their biological functions. Of course, the yeast methyltransferases contribute to gene activation by system is the most convenient for this type of methylation of protein substrates in addition to experiment, and such experiments have already shown histones must also be considered. The histone acetyl- that Hmt1/RMT1 and its methyltransferase activity are transferases p300, CBP, and p/CAF all have the ability important in RNA metabolism and for the nuclear- to acetylate speci®c non-histone proteins found in the cytoplasmic shuttling of speci®c RNA binding proteins transcription machinery, such as other coactivators, (Shen et al., 1998; McBride et al., 2000). However, the transcriptional activators, and basal transcription larger number of arginine-speci®c methyltransferase factors (Ogryzko et al., 1996; Gu and Roeder, 1997; proteins in mammalian cells than in yeast indicates that Imhof et al., 1997; Korzus et al., 1998; Chen et al., there may be more complex roles for these proteins in 1999b). By analogy, methyltransferases recruited to the higher eukaryotes. In mammalian cells antisense active promoters may have non-histone targets as well. technology and dominant negative mutants of CARM1 In fact, PRMT1 and the yeast Hmt1/RMT1 can and PRMT1 (if they can be developed) should prove methylate many RNA binding proteins which are known helpful in testing whether these proteins are required to be involved in various aspects of RNA processing, for transcriptional activation by nuclear receptors. transport, translation, and metabolism (Najbauer et al., Studies in dierentiating cell culture models, such as 1993; Lin et al., 1996; Gary and Clarke, 1998; Aletta et one which demonstrated a role for p160 coactivator al., 1998; Shen et al., 1998; Smith et al., 1999; Tang et al., GRIP1 in a developing myocyte system (Chen et al., 2000b; Nichols et al., 2000). The fact that transcriptional 2000b), may also prove useful. Genetic knockout and initiation and many post-transcriptional steps are known over-expression in transgenic mice will also help to to be coupled and coordinated (Monsalve et al., 2000) determine the roles of these proteins in hormone action suggests that some proteins currently classi®ed as and speci®c physiological processes; studies already transcriptional coactivators could actually function by performed with the p160 coactivators can serve as a facilitating post-transcriptional steps. For example, model for such studies (Xu et al., 1998, 2000). A mouse recruitment of PRMT1 to the promoter through its lacking functional PRMT1 has already been estab- contact with p160 coactivators could enhance reporter lished, and the phenotype of the homozygous null gene expression by methylation of (e.g.) histone H4, an mouse is embryonic lethal (Pawlak et al., 2000); but the RNA processing factor that is associated with the eect of this mutation on nuclear receptor function and transcription initiation complex, or both. In that regard, chromatin structure has not been examined. it is interesting that another coactivator, PGC-1 contains The fact that CARM1 and PRMT1 have arginine- RNA binding motifs and has been shown to help speci®c protein methyltransferase activities suggests stimulate and couple transcription initiation and sub- that protein methylation may be involved in their sequent splicing of the transcript (Monsalve et al., 2000). coactivator activities. In an initial genetic test of this In addition the yeast Hmt1/RMT1 has been shown to hypothesis, substitution of alanine for three highly interact genetically with proteins involved in RNA conserved residues (VLD) in the region of CARM1 processing and transport (Henry and Silver, 1996; Shen responsible for S-adenosylmethionine binding resulted et al., 1998), and the methyltransferase activity of Hmt1/ in loss of methyltransferase and coactivator activities, RMT1 is important for this function in yeast (McBride et thus supporting a role for protein methylation in the al., 2000). A number of unidenti®ed proteins in cell coactivator function of CARM1 (Chen et al., 1999a). extracts have been shown to be substrates for methyla- However, a recent X-ray crystal structure of the tion by speci®c or unspeci®ed methyltransferases (Lin et catalytic domain of PRMT3, which is closely related al., 1996; Tang et al., 1998; Frankel and Clarke, 1999; in sequence to that of CARM1, indicates that the VLD Pollack et al., 1999). Thus, it is likely that additional residues are probably involved in intramolecular methylation targets involved in regulation of transcrip- folding and thus may aect more than just S- tion or processes coupled to transcription remain to be adenosylmethionine binding (Zhang et al., 2000). Ideal identi®ed. mutations for testing the role of methylation in the As new methyltransferase substrates are identi®ed, it coactivator function of CARM1 should selectively will be desirable to address the physiological roles of eliminate binding of S-adenosylmethionine, protein their methylation. In order to do this, it will be

Oncogene Protein methylation in transcriptional regulation MR Stallcup 3019 necessary to know speci®c functions for the substrate reversible. Methylated histones can thus be used as proteins and to have speci®c assays to detect these substrates to search for enzymes that remove the methyl functions (e.g. cell free functional assays, transfection groups. An activity that removes epsilon-methyl groups assays in cultured cells, or gene replacement strategies from methyllysine of histones or from free methyllysine in living organisms). In that case, the importance of has been reported (Paik and Kim, 1973, 1974), but there methylation could be addressed by ®rst determining the is no evidence for an activity that can remove methyl methylation site(s) and modifying them genetically (e.g. groups from arginine residues of proteins. For other change lysine to arginine or arginine to lysine). The types of covalent modi®cation, such as acetylation and activities of the mutants which cannot be methylated phosphorylation, both the addition and the removal of could then be compared with the wild type protein in the modifying moiety are highly regulated. Therefore, it the available assay. is likely that the discovery of protein demethylases will also lead to additional signaling pathways which contribute to the complex and cooperative mechanisms Reversibility of protein methylation that regulate transcription and post-transcriptional Most steps in signal transduction pathways are readily nuclear events in gene expression. 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 Acknowledgments seems necessary to assume that the methylation is I thank Dr DW Aswad (University of California, CA, reversible or that the methylated protein can be USA), Dr BD Strahl (University of Virginia, VA, USA), eliminated or replaced by an unmethylated protein. and Dr H Ma (University of Southern California, CA, However, little is known about this process. The ®ndings USA) for critical comments on the manuscript. This work that at least some methylation of histones is dynamic was supported by US Public Health Service grant rather than static implies that methylation must be DK55274 from the National Institutes of Health.

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