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. 2009 Jan 22;23(4):425–433. doi: 10.1210/me.2008-0380

Minireview: Protein Arginine Methylation of Nonhistone Proteins in Transcriptional Regulation

Young-Ho Lee 1, Michael R Stallcup 1
PMCID: PMC2667706  PMID: 19164444

Abstract

Endocrine regulation frequently culminates in altered transcription of specific genes. The signal transduction pathways, which transmit the endocrine signal from cell surface to the transcription machinery, often involve posttranslational modifications of proteins. Although phosphorylation has been by far the most widely studied protein modification, recent studies have indicated important roles for other types of modification, including protein arginine methylation. Ten different protein arginine methyltransferase (PRMT) family members have been identified in mammalian cells, and numerous substrates are being identified for these PRMTs. Whereas major attention has been focused on the methylation of histones and its role in chromatin remodeling and transcriptional regulation, there are many nonhistone substrates methylated by PRMTs. This review primarily focuses on recent progress on the roles of the nonhistone protein methylation in transcription. Protein methylation of coactivators, transcription factors, and signal transducers, among other proteins, plays important roles in transcriptional regulation. Protein methylation may affect protein-protein interaction, protein-DNA or protein-RNA interaction, protein stability, subcellular localization, or enzymatic activity. Thus, protein arginine methylation is critical for regulation of transcription and potentially for various physiological/pathological processes.

This review focuses on the increasingly recognized roles of arginine methylation of non-histone proteins in regulating transcription.

Regulation of specific gene transcription by endocrine signals usually involves altered recruitment of transcriptional regulator proteins to the promoter/enhancer/silencer regions of target genes or alteration of the activity of proteins already associated with the gene. Frequently, these two mechanisms of gene regulation are accomplished by specific posttranslational modification of the proteins involved in transcriptional regulation. Such modifications alter protein function in specific ways. The roles of phosphorylation in transcriptional regulation have been extensively studied, but recently the importance of other types of protein modifications, including acetylation and methylation, have begun to be recognized. This review will focus on the roles of protein methylation, specifically arginine-directed methylation of nonhistone proteins, in transcriptional regulation. Because a substantial portion, but certainly not all, of the investigations have been conducted in the context of transcriptional regulation by nuclear receptors, the review will also focus, although not exclusively, on the nuclear receptors as a model system.

Protein methylation is one of the most abundant protein modifications. For example, about 2% of arginine residues were found to be dimethylated in total protein extracts from rat liver nuclei (1). Although protein methylation was first observed in the 1960s, the molecular roles for these modifications and the enzymes responsible remained obscure until recently (2). Protein methyltransferases transfer methyl groups (CH3-) from the S-adenosylmethionine methyl donor to specific methyl acceptors such as arginine, lysine, histidine, and carboxyl groups. Lysine residues can be modified by single, double, or triple methylation. Arginine can be monomethylated or dimethylated, and the latter can be asymmetric (both methyl groups on the same N atom at the end of the arginine side chain) or symmetrical (one methyl group on each of the two terminal N atoms), depending on the type of methyltransferase (3,4,5,6).

Histone tails are heavily modified by lysine and arginine methylation, in addition to other modifications such as acetylation, phosphorylation, and ubiquitylation. These histone tail modifications play important roles in chromatin remodeling and transcriptional regulation (7,8). Arginine methylation of histone tails has usually been associated with transcriptional activation, although its involvement with repression of transcription has also been documented in some cases. Lysine methylation of histone tails can contribute to either activation or repression of transcription, depending on position of methylation and the adjacent modifications. Because there are numerous reviews about histone methylation (5,7,9), this review will primarily focus on nonhistone protein methylation, especially the roles of protein arginine methylation in transcription in mammalian systems.

Transcriptional Regulation by Nuclear Receptors and Their Coregulators

Ligands of nuclear receptors function as allosteric regulators of receptor function by altering the shape of the receptor to which they bind (10,11). This ligand-triggered alteration in nuclear receptor shape regulates DNA binding, in the case of the steroid hormone receptors, and allows the DNA-bound receptor to bind coregulator proteins and thus recruit them to the target gene promoter. The coregulators play critical roles in regulating chromatin conformation and regulating the recruitment and activation of RNA polymerase II. Because more than 300 potential coregulators for nuclear receptors have been identified (http://www.nursa.org), these two rather simple-sounding tasks of coregulators are likely to be enormously complex. Some coregulators function by protein-protein interactions, e.g. by facilitating or inhibiting recruitment of other coregulators or specific components of the transcription machinery. Other coregulators are enzymes that catalyze posttranslational modifications to histones, nuclear receptors, other coregulators, and components of signal transduction pathways, among other targets (12,13,14). Thus, in addition to nuclear receptor ligands, posttranslational modifications of many (or perhaps all) components of the basal and regulatory transcription machinery also play major roles in nuclear receptor-mediated transcriptional regulation. These diverse posttranslational modifications can be catalyzed by the enzymatic coregulators that have been recruited by the nuclear receptor, in which case the modification may be an integral part of the process triggered by the nuclear receptor ligand. On the other hand, protein modifications can also result from activation of other cellular signal transduction pathways, in which case they may serve to modulate (i.e. enhance or inhibit) the outcome of the regulatory process triggered by the nuclear receptor ligand (15).

Protein Arginine Methyltransferases

Over the past 12 yr or so, 10 mammalian protein arginine methyltransferase (PRMT) family members have been identified, of which eight have been shown to catalyze methylation (5,6). The cDNAs encoding these enzymes were identified by a variety of methods, particularly yeast two-hybrid analysis and genetic screening. For example, the predominant methyltransferase in at least some mammalian cells, PRMT1 was identified as a binding partner for mitogen immediate-early response protein TIS21 and for leukemia-associated protein BTG1 in a yeast two-hybrid screen (16). PRMT2 was identified by sequence homology with PRMT1 (17). Coactivator-associated arginine methyltransferase 1 (CARM1)/PRMT4 was isolated as a binding partner for the C-terminal region of the nuclear receptor coactivator, glucocorticoid receptor-interacting protein 1 (18).

The 10 mammalian PRMTs fall into two predominant classes, based on the types of methylarginine products they produce (6). Type I enzymes (PRMT1, PRMT3, PRMT4/CARM1, PRMT6, and PRMT8) form monomethylarginine and asymmetric dimethylarginine, and type II enzymes (PRMT5, PRMT7, and FBXO11) form monomethylarginine and symmetric dimethylarginine. No activity has yet been demonstrated for PRMT2 and PRMT9.

PRMTs have conserved catalytic methyltransferase domains that are identifiable by a series of short conserved motifs that are important for binding of the methyl donor and for catalysis. In contrast, their N-terminal and C-terminal regions lack conservation in length or sequence (6,19). Some PRMTs are highly conserved from yeast to mammals. Three-dimensional structures of a few type I methyltransferase domains have been determined (19,20,21,22). These structures provided an understanding of S-adenosylmethionine binding and catalytic mechanism but only provided clues about protein substrate recognition surfaces and specificity, which still remain poorly understood. There have also been indications that the nonconserved N-terminal or C-terminal regions of PRMTs may play roles in determining their substrate specificity (21,22,23,24,25).

Subcellular locations vary among the PRMTs. For example, PRMT1 and CARM1 are both located in cytoplasm and nucleus, consistent with their roles in methylating histones and other transcription factors as well as cytoplasmic proteins such as RNA-binding proteins. In contrast, PRMT8 is mainly associated with the plasma membrane, suggesting unique roles for PRMT8 (6).

Protein Substrates for Arginine Methylation

The list of protein substrates for PRMTs is still growing. Histones are one class of well-defined substrates for PRMTs. For example, histone H3 is methylated by CARM1 on arginine residues 2, 17, and 26; histone H4 is methylated by PRMT1 on arginine 3; and other PRMTs also contribute to histone modification (5,6). These histone methylations are presumably involved in the recruitment of chromatin remodeling factors, but few specific molecular details have been elucidated.

PRMTs also methylate diverse nonhistone protein substrates including many RNA-binding proteins, signal transducers, DNA-binding transcriptional regulators, and transcriptional coregulators (2,5,6). Although there is some overlap in protein substrate specificity among the PRMTs, each enzyme has distinct substrates as well. Several of the enzymes (PRMT1, -3, -6, and -8) recognize glycine/arginine-rich (GAR) motifs (especially RGG repeats); but these PRMTs also methylate non-GAR substrates, and CARM1/PRMT4 only methylates non-GAR substrates with a specificity quite distinct from that of the other PRMTs. To date, no other motifs have been identified to explain the specific recognition of non-GAR substrates, suggesting that tertiary structure may be involved in recognition.

As discussed above, transcriptional coregulators are recruited by DNA-binding transcription factors, such as nuclear receptors, to promoters of target genes, where they are involved in chromatin remodeling and regulating recruitment and activation of RNA polymerase II (12,13,14). Some coregulators [such as the three p160 or steroid receptor coactivator (SRC) coactivators, the coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and the corepressor, receptor interacting protein 140 (RIP140)] bind directly to nuclear receptors and appear to serve as platforms or scaffolds for recruitment of other coregulators. For example, the p160 or SRC coactivators (SRC-1, glucocorticoid receptor interacting protein 1/transcriptional intermediary factor 2/SRC-2 and pCIP/SRC-3/amplified in breast cancer 1/activator of thyroid and retinoic acid receptor/receptor-associated coactivator 3) serve as scaffolds to recruit histone/protein acetyltransferases [e.g. cAMP response element-binding protein (CREB)-binding protein (CBP), p300, pCAF] and some of the PRMTs (e.g. PRMT1 and CARM1/PRMT4). These enzymatic coactivators are known to acetylate and methylate histones (as discussed above) in the local chromatin region where they are bound. However, as discussed below, they also modify other coregulators, other components of the transcription machinery, and even some nuclear receptors and members of other classes of DNA-binding transcription factors.

Transcriptional coregulators

A number of coregulators are methylated by PRMTs. It is worth noting that many, if not most, of the coregulators that function with nuclear receptors also function as coregulators for other classes of transcription factors. Therefore the implications of coregulator methylation events described below are likely to have broad significance within the transcription field.

Among the first coactivators shown to be methylated by PRMTs are p300 and CBP. The N-terminal KIX domain of p300 and CBP is one of several protein-protein interaction domains in these proteins. Methylation of several sites in and around the KIX domain by CARM1 was reported by two different groups, although the specific methylation sites and the specific functional consequences of the methylation were different (26,27). Xu et al. (26) found that the KIX region methylation regulates the interaction of CBP and p300 with the DNA-binding transcription factor CREB and thus alters the ability of CREB to activate transcription in response to cAMP. Chevillard-Briet et al. (27)found that CBP methylation in this region contributed to its ability to cooperate with the SRC coactivators. Methylation of a C-terminal region of p300 and CBP was shown to inhibit the critical interaction between p300 and the SRC coactivators (28). This interaction is critical for recruitment of p300 and CBP to nuclear receptor target genes, and thus the methylation of this region by CARM1 may be important for regulating coactivator complex assembly and disassembly.

The SRC/p160 coactivator family consists of three members that bind directly to nuclear receptors (as well as other classes of DNA-binding transcription factors) and serve as crucial scaffolds for recruitment of other coregulators. Two independent groups showed that one of the p160/SRC coactivators, p/CIP/AIB1/SRC-3 is methylated in its C-terminal region by CARM1. This methylation leads to the dissociation of CBP and CARM1 from SRC-3 and also reduces SRC-3 stability (29,30). Thus, methylation of SRC-3 by CARM1 may either regulate coactivator complex assembly or promote complex disassembly to maintain the rapid assembly-disassembly cycle of transcription complexes on the promoter (31).

RIP140, a ligand-dependent corepressor for nuclear receptors, is methylated by PRMT1 on three arginine residues. Methylation led to the suppression of the corepressor activity of RIP140, apparently by inhibiting RIP140 interaction with histone deacetylases and facilitating nuclear export of RIP140 (32).

PGC-1α serves as a coactivator for a number of nuclear receptors as well as other types of transcription factors, including nuclear respiratory factor 1. Expression of PGC-1α is induced by a variety of physiological stimuli that regulate metabolic activity, including exposure to cold, exercise, and fasting. As suggested by this regulatory pattern, PGC-1α regulates metabolic processes such as mitochondrial biogenesis, respiration, and gluconeogenesis. The C-terminal region of PGC-1α is methylated by PRMT1 in one or more sites within a glutamate- and arginine-rich region. Mutation of methylated arginine residues of PGC-1α or reduction of endogenous PRMT1 levels compromised PGC-1α coactivator activity and induction of target genes by PGC-1α overexpression (33). The protein-protein interactions affected by PGC-1α methylation have not been determined.

DNA-binding transcription factors

The variety of types of transcription factors for which arginine methylation has been demonstrated suggests that many more such modifications will be discovered eventually in this class of proteins and that protein methylation will prove to be a widespread transcriptional regulatory mechanism.

The signal transducer and activator of transcription (STAT) family of DNA-binding transcription factors is activated by a variety of signals, including growth factors and cytokines. STAT protein activation requires phosphorylation by tyrosine kinases and sometimes serine kinases in the cytoplasm, which leads to nuclear localization and binding as homo- or heterodimers to enhancer elements of immediate early response genes. PRMT1 was found to bind to the cytoplasmic domain of the interferon receptor, and reduction of cellular levels of PRMT1 reduced the antiviral and antiproliferative effects of interferon, suggesting a role for PRMT1 in the interferon response (34). Mowen et al. (35) showed that STAT1 is methylated by PRMT1 in vitro and in vivo and that inhibition of S-adenosylmethionine-dependent methylation inhibited the DNA binding activity of STAT1 and STAT1-mediated transcription. The inhibition of STAT1 DNA binding correlated with increased association of STAT1 with the inhibitory protein, protein inhibitor of activated STAT 1 (35,36).

Small mothers against decapentaplegic (SMADs) are DNA-binding transcription factors that modulate the activity of TGF-β ligands and receptors. There are three classes of SMAD molecules: R-SMAD (receptor-regulated SMADs 1, 2, 3, 5, and 9), Co-SMAD (the common heterodimer partner) SMAD4, and I-SMADs (inhibitory SMADs 6 and 7). R-SMADs are phosphorylated in the cytoplasm in response to TGF-β ligands, then form heterodimers with SMAD4, translocate into the nucleus, and bind specific enhancer elements. The I-SMADs, which inhibit signaling initiated by TGF-β ligands, are methylated in vivo by PRMT1, suggesting regulation of this pathway by arginine methylation (37). However, the functional significance of SMAD methylation has not been determined.

Estrogen receptor (ER)α, like other nuclear receptors, regulates transcription of specific genes in response to hormone, but hormone-activated ERα can also regulate cytoplasmic signaling independently of its nuclear actions. Le Romancer et al. (38) showed that Arg 260 of the ERα DNA-binding domain is rapidly and transiently methylated by PRMT1 in response to estrogen. Methylated ER forms a complex with Src kinase, phosphatidylinositol 3 kinase, and focal adhesion kinase, and this complex regulates hormone-mediated proliferation and migration of breast cancer cells. The methylated form of ER is predominantly cytoplasmic, and hypermethylation of ER is observed in a subset of breast tumors.

Methylation of the DNA-binding domain of orphan nuclear receptor hepatocyte nuclear factor 4 by PRMT1 enhances DNA binding, whereas PRMT1 also contributes to transcriptional activation in the more classical manner, by methylation of histone H3 at arginine 4 (39).

RUNX1 is a transcription factor required for hematopoiesis, and its gene is often involved in mutations and chromosomal translocations that contribute to acute leukemia. RUNX1 is methylated in vivo by PRMT1 on arginine residues 206 and 210, and PRMT1 serves as a transcriptional coactivator for RUNX1. The methylation by PRMT1 abrogates association of RUNX1 with corepressor SIN3A and thus plays a role in transcriptional activation by RUNX1. Arginine-methylated RUNX1 is recruited to the promoters of two RUNX1 target genes, CD41 and PU.1. However, the leukemia-associated RUNX1-ETO fusion protein lacks these arginine methylation sites, causing constitutive corepressor binding (40).

Transcription elongation factors

SPT5 and binding partner SPT4 regulate transcription elongation. SPT5 is methylated by both PRMT1 and PRMT5 in vitro and in vivo. Activation of transcription of specific genes was found to increase association of SPT5 with the activated genes but decrease PRMT1 and PRMT5 occupancy of the same genes. Mutating the methylation sites of SPT5 affected its association with RNA polymerase II and its ability to stimulate transcription elongation in vitro (41).

Another transcription elongation factor, TFIIF-associating component of CTD phosphatase 1 (FCP1), is a phosphatase for the carboxyl-terminal domain of the large subunit of RNA polymerase II. Amente et al. (42) showed that FCP1 interacts with PRMT5 and is methylated by PRMT5 in vitro and in vivo. However, the roles of arginine methylation in the function of FCP1 are not known.

Methyl-DNA-binding protein (MBD)

DNA methylation is an important epigenetic mechanism for maintaining repressive chromatin structure. Methylated DNA is recognized by MBD proteins. MBD proteins recruit histone deacetylases and thus assemble a transcriptional repression complex. MBD2 is methylated by PRMT1 and PRMT5 within a glycine/arginine-rich region in vitro and in vivo. Arginine methylation of MBD2 reduced the ability of MBD2 to bind methylated DNA and histone deacetylases and thus inhibited the formation of a transcriptional repression complex on methylated DNA (43).

DNA damage response proteins

MRE11, a key enzyme in DNA double-strand break repair and genome stability, has a glycine-arginine-rich (GAR) motif that is methylated by PRMT1. Although reduced methylation did not prevent MRE11 from forming a complex with two other critical DNA repair proteins, RAD50 and NBS1, it impaired the exonuclease activity of MRE11 and its relocation from PML bodies to sites of DNA damage (44,45,46).

p53-binding protein 1 (53BP1) is also a protein factor rapidly recruited to sites of DNA double-strand breaks. Similar to MRE11, 53BP1 also contains a GAR motif that is arginine methylated by PRMT1. Amino acid substitution of the arginines within the GAR motif of 53BP1 or treatment of cells with a general methyltranferase inhibitor abrogated binding of 53BP1 to single and double-stranded DNA and relocalization of 53BP1 to DNA damage sites (47).

RNA-binding proteins

Thus far, one of the most frequently identified types of protein targets for arginine methylation is RNA-binding proteins. RNA-binding proteins are involved in processing, folding, stabilization, localization, and translation of RNAs. Methylation of these proteins has been found to alter diverse functions, including RNA-protein interactions, subcellular localization of proteins, protein-protein interactions that lead to ribonucleoprotein complex formation, and mRNA processing. Because these methylation events are less directly related to transcriptional regulation and have been reviewed thoroughly (4,5,6,48), they will not be discussed in detail here. However, in one recent transcription-related study, arginine methylation of the RNA-binding protein, heterogenous nuclear ribonucleoprotein K, was found to increase in response to UV irradiation, and inhibition of methylation by use of a general S-adenosylmethionine-dependent methylation inhibitor or reduction in PRMT1 levels reduced p53 transcriptional activity, expression of the p53 target gene p21 in response to UV irradiation, and recruitment of heterogenous nuclear ribonucleoprotein K to the p21 promoter (49).

Specific Mechanisms of Transcriptional Regulation by Protein Arginine Methylation

As described above and further discussed below, protein arginine methylation can influence transcription by a variety of molecular mechanisms. The great majority of the cases probably involve either positive or negative effects on the interaction of the methylated proteins with other molecules (e.g. proteins, DNA, or RNA). However, the end result of these altered intermolecular interactions can involve effects on subcellular localization of proteins, on protein or RNA stability, or on the enzymatic activity of proteins.

Protein-protein interactions

Although methylation of arginine residues does not alter the charge of proteins, it can influence the hydrogen bonding and local hydrophobicity, both of which can affect intramolecular or intermolecular interactions. In most cases reported to date, arginine methylation has inhibited specific protein-protein interactions, but a few cases in which arginine methylation enhanced protein-protein interactions have also been reported. If methylation occurs at the interaction interface, it can have a direct effect on the interaction. However, indirect allosteric effects are also possible.

One example of inhibitory effects of arginine methylation on protein-protein interactions involves the histone H3 N-terminal tail. Histone tail modifications constitute a so-called histone code, and the various posttranslational modifications enhance or inhibit binding of specific proteins involved in chromatin remodeling and transcriptional regulation (7,8). A number of proteins have been identified which use tudor, PHD finger, or WD40 repeat domains to bind preferentially to histone H3, which has been trimethylated at lysine 4, a mark associated with active chromatin. Iberg et al. (50) demonstrated that the binding of several such proteins to lysine 4-methylated histone H3 is inhibited if arginine 2 of histone H3 contains asymmetric dimethylarginine. PRMT6 was identified as the enzyme responsible for at least the majority of this methyl mark in cells, and reduced levels of PRMT6 led to altered expression of several genes known to be regulated by the proteins that bind to trimethylated lysine 4 on histone H3. Other examples (discussed in the previous section of this review) of negative regulation of protein-protein interactions by arginine methylation include binding of p300 to p160/SRC coactivators (28), interaction of SRC-3 with CARM1 (30), binding of STAT1 to protein inhibitor of activated STAT 1 (35,36), binding of RUNX1 to SIN3A (40), and binding of RIP140 to histone deacetylases (32).

Only a few cases in which arginine methylation promotes protein-protein interactions have been defined. Cote and Richard (51) demonstrated that tudor domains of spinal muscular atrophy (SMN), splicing factor 30 kDa (SPF30), and tudor domain-containing 3 (TDRD3) proteins bind preferentially to methylated GAR motifs and to a number of cellular proteins that contain symmetric dimethylarginine. Cheng et al. (52) demonstrated that methylation of splicing factor CA150 by CARM1 (which generates asymmetric dimethylarginine) was required for binding of CA150 to the tudor domain of the spinal muscular atrophy protein. Thus, a subset of tudor domain-containing proteins can bind preferentially to arginine-methylated proteins.

Protein-DNA interactions

Xenopus ILF3, a DNA-binding transcription factor that also binds RNA and influences posttranscriptional events, can be methylated by PRMT1. This methylation inhibits DNA binding but not RNA binding, and overexpression of PRMT1 in Xenopus embryos caused reduced expression of an ILF3 target gene (53). Other examples, discussed in the previous section, involve DNA binding by 53BP1 (47), hepatocyte nuclear factor 4 (39), and MBD2 (43).

Protein subcellular localization

In yeast the PRMT1 homolog Hmt1p methylates a number of RNA-binding proteins, including Npl3p and Hrp1p, and thereby facilitates their nuclear export (54). Arginine methylation of RNA helicase A is required for its nuclear import via a classical import mechanism involving karyopherin β (55).

Protein stability

As discussed above, methylation of the C-terminal glutamine-rich region of SRC-3 by CARM1 enhances turnover of the protein (29).

Enzymatic activities of proteins

Many protein factors involved in transcription have enzymatic activities, such as kinase, acetyltransferase, and methyltransferase, which are very important for the regulation of transcription. Thus far there are very few examples of protein methylation causing changes in enzymatic activity. One example, discussed above, involves the exonuclease activity of MRE 11 (45).

RNA stability

RNA-binding proteins are a major class of substrates for arginine methylation (48). Proteins such as HuR and HuD bind to sequences in the 3′-untranslated regions of mRNAs, which are rich in adenosine and uridine, and thereby stabilize a number of otherwise unstable mRNAs. HuR is methylated by CARM1 in response to lipopolysaccharide exposure of macrophage cells (56). Because lipopolysaccharide is also known to stabilize TNFα mRNA through an HuR-dependent mechanism, it was proposed that HuR methylation might alter TNFα mRNA stability by either enhancing RNA binding affinity of HuR or altering its subcellular localization to increase its cytoplasmic level.

Regulation of Protein Arginine Methylation Levels

As with other types of posttranslational modifications, it seems very unlikely that arginine methylation of proteins is a constitutive modification that occurs automatically to newly synthesized substrate proteins and remains in place until the degradation of the protein. Although the pathways regulating protein arginine methylation are only beginning to be elucidated, specific examples suggest that there are several types of regulatory mechanisms. First, PRMT activity can be regulated by direct protein-protein interactions. Perhaps the first example reported was the stimulation of PRMT1 activity by interaction with either of two related proteins, TIS21 and BTG1 (16). In contrast, PRMT3 activity is inhibited by interaction with the tumor suppressor protein DAL-1 (57). PRMT5 can be found in various nuclear and cytoplasmic complexes (48). Association of PRMT5 with the chromatin remodeling adenosine triphosphatases, Brg1 and Brm1, enhances its methyltransferase activity (58). The substrate specificity of CARM1 is altered by association of CARM1 with a large ATP-dependent chromatin-remodeling complex that includes the adenosine triphosphatase subunit Brg1. Whereas free CARM1 prefers to methylate free histone H3 over H3 in nucleosomal substrates, CARM1 associated with the large complex prefers the nucleosomal substrate (59). PRMT7 activity is stimulated by interaction with the testis-specific chromosomal protein CTCFL/BORIS, and these proteins have been implicated in the imprinting of the Igf2/H19 locus during germ cell development (60).

Second, posttranslational modifications may regulate PRMT activity. Although some PRMTs have been found to self-methylate (61), the functional significance of these modifications in unknown. Recently, one example of phosphorylation-mediated regulation of PRMT activity has been reported. Homodimerization or homooligomerization of some of the PRMTs, including PRMT1, yeast Hmt1, and CARM1, is required for enzymatic activity (20,62,63). This suggests that regulation of dimerization or oligomerization could be used to regulate methyltransferase activity. CARM1 phosphorylation by an unknown kinase can be detected in a cell cycle-specific manner during G2/M phase. A serine-to-glutamate mutation intended to mimic phosphorylation exhibited reduced binding of S-adenosylmethionine, reduced methyltransferase activity, and reduced coactivator function with the ER (64).

Third, protein arginine methylation can be modulated by regulation of substrate accessibility. For example, PRMTs and other histone-modifying enzymes do not catalyze uniform histone modifications throughout the genome. Instead, when DNA-binding transcription factors activate transcription of specific genes, they recruit a host of coregulator proteins specifically to the target genes. CARM1 and PRMT1 (and presumably other PRMTs) are recruited to target genes by direct or indirect interaction with nuclear receptors and catalyze histone methylation locally within the chromatin (65,66,67). Similarly, PRMT1 is recruited to target gene promoters by the transcription factor Yin Yang 1 (68). It is easy to envision other scenarios where the access of PRMT to substrate proteins will be regulated by controlling the subcellular localization of PRMT or substrate protein.

Fourth, arginine methylation can theoretically be reversed by removal of the methyl group(s). Recently, such an enzyme has been reported (69). JMJD6 belongs to the family of Jumonji domain-containing proteins, several of which have been found to function as protein lysine demethylases, using Fe(ii) and α-ketoglutarate as cofactors (70,71). Another relevant enzyme is protein arginine deiminase (PAD)4 or PADI4, which converts arginine in proteins to citrulline by deimination, i.e. removal of an amino group from the guanidino side chain of arginine (72,73). Deimination effectively prevents methylation by PRMTs. This enzyme not only works on histones at potential arginine methylation sites (72,73), but also on p300, targeting at least one site that can be methylated by CARM1 (28). This enzyme was also shown initially to convert monomethylarginine (but not dimethylarginine) to citrulline; however, subsequent work indicated that monomethylarginine is a poor substrate for PAD4 (74,75). Nevertheless, the deimination of potential arginine methylation sites represents a relevant mechanism for regulating protein arginine methylation. It is interesting to speculate whether citrulline in histones and nonhistone proteins can be directly converted back to arginine by a currently unknown aminotransferase, or whether these citrullinated proteins are simply replaced by turnover and resynthesis.

Future Directions and Technical Challenges

Progress to date has undoubtedly uncovered only a small portion of the roles of arginine methylation in regulation of protein function in physiological processes. Investigation of the involvement of protein arginine methylation in human diseases has also begun (48,76) but will undoubtedly expand dramatically along with basic knowledge of protein methylation. In addition, although most of the mammalian PRMTs are probably known at this point, work on the identities and roles of demethylases and deiminases in regulating protein arginine methylation has barely begun and is an area that needs particular emphasis in the future.

Typical strategies for investigating protein arginine methylation

A typical strategy might begin by first demonstrating that recombinant PRMT can methylate a recombinant protein substrate in vitro, followed by deletion mapping to identify the methylated region. Mass spectrometry has been used successfully in some cases to identify the specific arginine residues that are methylated, but in other cases substitution of candidate arginine residues has been used. Of course, the latter method only shows that the mutated arginine residue is required for methylation; it could be the methylation site or could be simply important for recognition by the PRMT. In any case, methylation of the protein and the proposed site of methylation must be confirmed in vivo. Again, mass spectrometry would be the ideal method, but achieving success with proteins isolated from cells is much more challenging than analysis of a recombinant protein that has been methylated in vitro. An alternative, albeit potentially time-consuming, method that has been successful is to use antibodies that preferentially recognize the methylated vs. the unmethylated proteins. If commercially available antibodies against methylarginine do not detect the methylated protein (see further discussion below), it will be necessary to generate an antibody specific for the methylated protein of interest, using a synthetic peptide representing the methylated site of the protein. If a signal is obtained with such an antibody (e.g. by Western blot) in analyzing cell extracts, then it is important to demonstrate the specificity of the signal for the methylated vs. the unmethylated protein. This can be accomplished in a variety of ways by showing that the signal obtained with the methylarginine-specific antibody can be modulated without altering the level of the protein substrate. This can be achieved by modulating the cellular level of the PRMT responsible for the methylation; inhibiting methylation with compounds that block S-adenosylmethionine-dependent methylation; or comparing methylated vs. unmethylated peptides representing the methylation site as competitors to block the signal. To investigate function, point mutants lacking the methylation site are useful. In particular, it is useful to modulate the cellular level of the PRMT and test the effect of this on the function of the wild type vs. the methylation-deficient mutant. Comparison of wild type and mutant is useful to determine whether the effect of PRMT modulation on the function of the protein substrate is direct or indirect. If PRMT modulation has a direct effect, we would expect that it affects the function of the wild-type protein but not that of the unmethylatable mutant protein.

Need for new methods and technologies

Despite its promise, protein methylation has been technologically a challenging field of investigation, because of a lack of sophisticated research tools. For these reasons many investigations have used relatively laborious methods to identify specific sites of methylation in proteins, demonstrate that those same modifications occur in vivo, and investigate the physiological roles of these modifications. Development of new investigative tools and technology would help to speed up progress for investigators in this field, who otherwise will have to continue using laborious strategies such as those outlined above. Several types of desirable new tools and methods can be envisioned. First, because no reliable consensus sequence, aside from the GAR motif, has been identified for PRMTs, identification of new PRMT protein substrates and potential methylation sites within those substrates is very difficult. Fortunately, there have been recent advances in development of new high-throughput methods for detecting substrates for PRMTs (6,52,77). Second, improved mass spectrometry methods for identification of methylated proteins from cell extracts and determination of the precise methylation sites would be extremely helpful. There are several examples of recent advances in this area (78,79). However, identification of specific methylation sites by mass spectrometry is probably still not a routinely offered service of most mass spectrometry core facilities and may require collaboration with an experienced mass spectrometry laboratory to achieve success. Third, inhibitors specific to PRMTs or even individual PRMTs would be a dramatic improvement over the current compounds, which inhibit all S-adenosylmethionine-dependent methylation (of proteins, DNA, and small molecules) and thus make interpretation of results very difficult. In addition, inhibitors of demethylases would be extremely useful. Although much work still remains to be done to achieve compounds of the desired efficacy and specificity, there has been recent progress with PRMT-specific inhibitors (80,81,82) and PAD demethylase inhibitors (83,84,85). In addition to chemical inhibitors, use of RNA interference to reduce cellular levels of a specific enzyme has been, and will continue to be, an extremely useful and powerful strategy for investigation of physiological roles of PRMTs (86); this is particularly true because some functions of PRMTs involve protein-protein interactions rather than the methyltransferase activity (87). Fourth, availability of antibodies that recognize methylated arginine in the context of any protein would greatly simplify the identification of methylated proteins in vivo, because they would alleviate the need to make new antibodies for each new methylated protein discovered. Currently, antibodies designed to recognize any protein containing symmetric dimethylarginine or asymmetric dimethylarginine are available commercially. However, in our experience these antibodies, although useful for some methylated proteins, are still context specific and thus fail to recognize many arginine-methylated proteins.

Investigation into the physiological roles of protein arginine methylation has expanded very rapidly in the past decade or so. The many dramatic discoveries that have been made illustrate the importance of protein arginine methylation as a novel method for regulating the diverse functions of proteins and create a strong argument for continued and enhanced investigation in this field.

Footnotes

This work was supported by National Institutes of Health Grants DK43093 and DK55274 (to M.R.S.).

Disclosure Summary: Y.L. has nothing to declare. M.S. has previously consulted for Millipore Corp. (Upstate Biotechnology, Inc.).

First Published Online January 22, 2009

Abbreviations: 53BP1, p53-Binding protein 1; CARM1, coactivator-associated arginine methyltransferase 1; CBP, CREB-binding protein; CREB, cAMP response element-binding protein; ER, estrogen receptor; FCP1, TFIIF-associating component of CTD phosphatase 1; GAR, glycine/arginine-rich; MBD, methyl-DNA binding domain; PAD, protein arginine deiminase; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; PRMT, protein arginine methyltransferase; RIP140, receptor interacting protein 140; SMADs, small mothers against decapentaplegics; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription.

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