Role of Protein Methylation in Regulation of Transcription

David Y. Lee, Catherine Teyssier, Brian D. Strahl, and Michael R. Stallcup Departments of Biochemistry and Molecular Biology (D.Y.L., M.R.S.) and Pathology (C.T., M.R.S.), University of Southern California, Los Angeles, California 90089; and Department of Biochemistry and Biophysics (B.D.S.), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

In the last few years, the discovery of lysine and arginine noubiquitylation; combinations of these modifications cooper- methylation in histones and other proteins and the enzymes ate to regulate chromatin structure and transcription by stim- that carry out these posttranslational modifications has ulating or inhibiting binding of specific proteins. Although added a new dimension to the signal transduction field. In lysine methylation has thus far been observed almost exclu- particular, there has been a huge surge in our understanding sively on histones, arginine methylation has been observed on a of how methylation of nucleosomal histones at specific lysine or variety of other proteins associated with gene regulation, arginine residues affects chromatin conformations and either including DNA-binding transcriptional activators, transcrip- facilitates or inhibits transcription from neighboring genes. It tional coactivators, and many RNA binding proteins involved in appears that the responsible methyltransferases can be targeted RNA processing, transport, and stability. Thus, lysine and argi- in some cases to specific genes and in other cases to broader nine methylation of proteins, like many other types of posttrans- regions of euchromatin or heterochromatin. Methylation of lational modifications, are regulated steps of many specific sig- histones is mechanistically linked to other types of histone naling pathways. (Endocrine Reviews 26: 147–170, 2005) modifications, such as acetylation, phosphorylation, and mo-

I. Introduction B. Are arginine and lysine methylation reversible or stable II. Lysine Methylation marks? A. Enzymes C. Future directions B. Overview of histone modifications C. Functional implications of methylation of individual lysine residues of histones I. Introduction D. Consequences of histone lysine methylation E. Lysine methylation of a basal transcription factor ROTEIN METHYLATION CAN occur on arginine, ly- III. Arginine Methylation P sine, histidine, proline, and carboxyl groups. Studies A. Enzymes within the last decade have identified a wide variety of B. Effects of arginine methylation on protein function posttranslational modifications that occur on histones and C. Regulation of arginine methyltransferase activity other proteins involved in the regulation of transcription, IV. Conclusions and Unanswered Questions including lysine- and arginine-specific methylation (1, 2). A. Protein methylation and endocrinology This review will discuss the roles of lysine and arginine methylation in regulating gene expression at a variety of levels, but will emphasize the role of histone methylation in First Published Online October 12, 2004 Abbreviations: AdoHcy, S-Adenosylhomocysteine; AdoMet, S- the regulation of chromatin structure and transcription. Be- adenosyl-l-methionine; CARM1, coactivator-associated arginine fore 1999, evidence suggested that protein arginine methyl- methyltransferase-1; CBP, CREB-binding protein; CREB, cAMP re- ation is involved in various signaling pathways (3) and that sponse element binding protein; CTD, C-terminal domain; E(z), en- the methylation of some RNA binding proteins is involved hancer of zeste; GRIP1, glucocorticoid receptor-interacting protein-1; in their nuclear-cytoplasmic shuttling (4). Similarly, lysine HMT, histone methyltransferase; hnRNP, heterogeneous nuclear ribonucleoprotein; HP1, heterochromatin protein 1; LPS, lipopolysaccha- methylation of histones had been extensively documented, ride; mAM, a murine activating transcription factor-associated modu- but the function of this modification on histones remained lator; NGF, nerve growth factor; NuRD, nucleosome remodeling and elusive (5). In 1999, two papers provided compelling evi- deacetylase; Pc, polycomb; pCAF, p300/CBP-associated factor; PEV, dence that lysine and arginine methylation of histones func- position effect variegation; PIAS1, protein inhibitor of activated STAT1; PRC1, polycomb repressive complex-1; PRMT, protein arginine methyl- tions in the process of gene transcription (6, 7). Since then, an transferase; Rb, retinoblastoma; rDNA, ribosomal DNA; RNAi, RNA explosion of new information on this topic, as well as the interference; RNA pol II, RNA polymerase II; SET, su(var), Enhancer of development of a new field of chromatin biology, has zeste, trithorax; shRNA, short heterochromatic RNA; SMN, survival of occurred. motor neuron; snRNPs, small nuclear ribonucleoprotein particles; STAT, Chromatin structure plays an integral role in the control of signal transducer and activator of transcription; TRR, TRX-related; TRX, trithorax; Ubx, ultrabithorax. gene expression. The basic repeating unit of chromatin is the Endocrine Reviews is published bimonthly by The Endocrine Society nucleosome, in which 146 bp of DNA wraps around an (http://www.endo-society.org), the foremost professional society serv- octamer of core histones, consisting of pairs of H3, H4, H2A, ing the endocrine community. and H2B (8). N-terminal tails of histones protrude out of the

147 148 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription nucleosome and are subject to a variety of posttranslational 2. Mechanism and regulation of enzymatic activity and substrate modifications such as acetylation, phosphorylation, ubiqui- specificity. HMTs display exquisite substrate specificity, not tylation, and lysine and arginine methylation. Acetylation only for specific lysine residues of specific histones but also was the first of these modifications to be linked with active for free histones vs. nucleosomes. Dot1, Set2, and PR-Set7/ transcription, and subsequently phosphorylation of histone Set8 can only methylate histone tails presented in the context H3 was found to cooperate with acetylation in transcriptional of nucleosomes (24–27), whereas other HMTs prefer free activation (9, 10). There are many sites of lysine and arginine histones or can methylate tails from both free histones and methylation in histones, and they play a variety of important nucleosomes. Those that prefer free histones over nucleo- and, in some cases, essential roles in regulating chromatin somes may require additional subunits to allow methylation structure and gene transcription. Some histone methylation of nucleosomes, as was shown previously for the yeast hi- events, e.g., methylation of Lys-4 and Arg-17 of histone H3 stone acetyltransferase Gcn5 when tested by itself or as part and Arg-3 of histone H4, have also been associated with of the SAGA complex (28). transcription activation; in contrast, methylation of H3 Lys-9 Although histones are by far the predominant substrates has been correlated with gene silencing (11, 12). Although identified for HMTs, a few nonhistone substrates were pre- many correlations of specific histone modifications with ac- viously identified: calmodulin, cytochrome c, and ribulose- tive or inactive chromatin are generally valid, exceptions do 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from exist; it is now widely held that individual histone modifi- plants (19). In fact, the idea that Suv39h1 might have HMT cations may not constitute clear signals by themselves, but activity arose from the sequence homology among the rather multiple modifications probably function together as Suv39h1 SET domain and other proteins with SET domains, part of a histone code which states that sequential or con- including plant Rubisco large subunit methyltransferases, current combinations of modifications constitute signals that which were known to methylate Lys-14 of Rubisco (29). Thus, are read by other proteins (1, 2, 13). it would not be surprising to find a whole range of other Although histones (specifically H3 and H4) have so far proteins that are methylated on lysine residues; a similar been the stars of the saga of protein methylation in tran- experimental trend occurred with protein acetylation that scriptional regulation, recent work has shown that methyl- was initially characterized primarily on histones but later ation regulates the activities of an increasing list of other recognized as a posttranslational modification for many non- components of the transcription machinery. In addition to histone proteins (30–33). addressing how methylation of nonhistone proteins contributes to transcriptional regulation, we will also briefly discuss B. Overview of histone modifications how methylation of a variety of protein substrates contributes to regulation of various posttranscriptional levels of Histones were long regarded as a passive packaging struc- gene regulation and to the regulation of various cellular ture for DNA. Of course, now it is widely recognized that signal transduction pathways. histones play a dynamic role in controlling chromatin structure and transcription. There are apparently many different states of chromatin compaction. The more compact chroma- II. Lysine Methylation tin, called heterochromatin, generally is characterized by late A. Enzymes replication during S phase (due to its highly condensed nature), low gene density, and repetitive DNA sequences (34, 1. Classification of histone lysine methyltransferases and their 35). Euchromatin has a more open structure and contains histone substrates. Histone lysine methylation occurs on his- genes that are active or potentially active. It is now clear that tone H3 at lysines 4, 9, 14, 27, 36, and 79 and on histone H4 histone lysine methylation plays a major role in regulating at lysines 20 and 59 (1, 12, 14–16). Many of the enzymes that the state of chromatin compaction, and thus the establish- modify these particular residues have been isolated and char- ment and maintenance of heterochromatic and euchromatic acterized (Fig. 1A), and crystal structures have been deter- regions in chromatin. In addition, histone lysine methylation mined for some of them (17–23). All of the lysine-specific also apparently plays central roles in regulating activation histone methyltransferases (HMTs) except Dot1 share a SET and repression of gene transcription within euchromatin. [Su(var), Enhancer of zeste, trithorax] domain that is respon- In general, methylation of histone H3 at Lys-4, 36, and 79 sible for catalysis and binding of cofactor S-adenosyl-l- is correlated with euchromatin and transcriptional activa- methionine (AdoMet). HMTs then add one or more methyl tion, whereas methylation of histone H3 at Lys-9 and 27 and groups to the ⑀-amino group of lysine residues, resulting in histone H4 at Lys-20 is associated with heterochromatin and mono-, di-, or trimethylated lysine (Fig. 1B). Methylation of transcriptional repression (Figs. 2-4). However, this is not lysine residues does not change the net positive charge but always the case, and it should be noted that the specific progressively increases the bulk and hydrophobicity and functions of various histone modifications are still under may disrupt intra- or intermolecular hydrogen-bond inter- intensive investigation; thus the information summarized actions of the ⑀-amino group or create new sites for proteins here should be considered as a progress report with the that bind preferentially to the methylated protein. N-C bonds understanding that many new insights are yet to come. Fur- of methyllysine are very stable, and so far no demethylases thermore, the histone code appears to have some variations have been discovered, leading to questions about the revers- in certain organisms, particularly in budding yeasts. For ibility of this modification. These issues will be discussed example, methylation of histone H3 at Lys-9, the Suv39h1 further in Section IV. class of HMTs responsible for that modification, and the Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 149

FIG. 1. Arg and Lys methylation of histones. A, Methylation sites of histones H3 and H4. The N-terminal amino acid sequences of histones H3 and H4 are shown along with positions of specific methylation sites and some of the HMT and PRMT enzymes responsible for methylation at each site. Arg methylation is shown above the sequence, and Lys methylation below. The names of the three enzymes for which the SET domains of Lys HMTs were named are indicated on the left. Lys residues known to be acetylated are shown in red, and Ser residues known to be phosphorylated are shown in blue. B, Lys methylation. The structures of mono-, di-, and trimethyllysine are shown. C, Arg methylation. Structures of monomethyl-, asymmetric dimethyl-, and symmetric dimethylarginine are shown, along with the type of PRMT enzyme responsible for synthesizing each species. heterochromatin protein 1 (HP1) that binds to histone H3 in higher eukaryotes (7, 26, 27, 36). Instead, S. cerevisiae uses methylated at Lys-9 do not exist in Saccharomyces cerevisiae, H3 Lys-4 and Lys-79 methylation to limit the extent of silent which also lacks the large blocks of heterochromatin found domains with the help of Sir silencing proteins (24). One 150 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

FIG. 2. Examples of transcriptional regulation mechanisms by histone lysine methylation. A, The molecules responsible for or involved in recruitment of the HMT. There are both global and gene-specific mechanisms for recruiting HMTs to broad regions of heterochromatin and euchromatin or to specific genes, respectively. Molecules responsible for global recruitment (see text) include: shRNA or short heterochromatic RNA, short double-stranded RNA transcribed from centromeric heterochromatin; Xist, RNA transcribed from the Xist gene and bound to the inactive X chromosome; and Paf1, the Paf1 protein complex associated with RNA pol II. DNA-binding transcriptional activator proteins or cofactor (coactivator or corepressor) proteins that can recruit specific HMTs to specific genes (see text) include: PRDI-BF1 transcription factor; Rb, the retinoblastoma protein, a corepressor that is recruited by specific transcription factors; GAGA transcription factors; EcR, the Drosophila ecdysone receptor, a nuclear hormone receptor family transcriptional activator protein. In addition, the Ser-5-phosphorylated CTD of RNA Pol II and monoubiquitylated histone H2B can recruit specific HMTs to active genes. B, HMT enzyme responsible for the modification. C, Specific histone H3 residue methylated. D, Protein that binds or is prevented from binding by the specific histone H3 methylation. Arrowheads indicate positive effects, whereas lines with flat ends indicate inhibition. HAT, p300 histone acetyltransferase. E, Effect on transcription. mam, Mammalian; d, Drosophila; sc, S. cerevisiae; sp, S. pombe.

FIG. 3. Distribution of histone H3 Lys methylation and key proteins responsible for formation and maintenance of heterochromatin and euchromatin. Similar patterns of Lys methylation have been found in fission yeast (S. pombe), budding yeast (S. cerevisiae), and humans (H. sapiens). Centromeric heterochromatin in the indicated organisms contains HP1 proteins, which bind to histone H3 methylated at Lys-9, and also contains the Lys-9 HMT Suv39h1, which is recruited by shRNA and by HP1. These findings suggest a mechanism for spreading of heterochromatin structure. Lys-4 and Lys-79 methylation in euchromatic regions is thought to limit the spreading of heterochromatin by facilitating the binding of proteins that promote euchromatin structure and inhibiting the binding of proteins that promote heterochromatin spreading (see Figs. 2 and 4). The centromeric and telomeric regions of budding yeast do not have the dense heterochromatin characteristic of fission yeast and more complex eukaryotes, but the chromatin in budding yeast telomeric regions shares some characteristics of the heterochromatin structure of the other organisms, including the presence of Sir proteins in the telomeric regions. The fact that different proteins bind to telomeric vs. centromeric heterochromatin suggests that there are different types of heterochromatin regulated by different mechanisms. Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 151

FIG. 4. Regulation of Lys methylation of histone H3 by monoubiquitylation of histone H2B, by the Paf1 protein complex, and by phosphorylation of the CTD of RNA pol II. Lys-4, Lys-36, and Lys-79 methylation of histone H3, which are generally associated with active transcription, are regulated by a series of prior events (shown on the left) and promote active transcription by influencing the binding or activity of specific proteins on histone H3 (shown on the right). Arrowheads indicate positive effects, and lines with flat ends represent negative effects. Enzymes or proteins responsible for the positive effects are in boxes associated with the arrows. Monoubiquitylation of histone H2B at Lys-123 by Rad6 (ubiquitin E2 ligase) and Bre1/Lge1 (ubiquitin E3 ligase) leads to methylation of histone H3 at Lys-4 and Lys-79. Set1 preferentially associates with the CTD of RNA pol II, which is phosphorylated at Ser-5. Set2 prefers to bind the CTD of RNA pol II phosphorylated at Ser-2. The Paf1 complex contributes to the recruitment of several of the key enzymes. Lys-4 methylation promotes remodeling of chromatin to the active state by attracting p300 histone acetyltransferases (HAT) and coactivator Isw1p and by inhibiting the binding of the NuRD corepressor complex. Lys-79 methylation in euchromatin inhibits the binding of repressive Sir proteins. The mechanism by which Lys-36 methylation contributes to active transcription is unknown. reason for this difference may be that the budding yeasts vation and repression, either in different regulatory situa- have very little repetitive DNA compared with higher eu- tions or due to differences in the histone code among karyotes. Thus, the general correlation between each histone different species. Below, for each modification we discuss the modification and its function may not be universally true evidence for its involvement in activation or repression or among all species, and caution should be used about inter- both. The downstream consequences of Lys methylation will pretation and generalization of findings across wide evolu- be discussed in Section II.D. tionary gaps. As will become evident below, the degree of methylation 1. H3 Lys-4 methylation of a specific histone lysine residue (i.e., mono-, di-, or tri- a. Activation methylation) may vary according to the context in which it occurs or the specific enzyme that makes the modification. i. Global chromatin structure. At the global chromatin level, Much work is still needed to completely elucidate the overwhelming evidence supports association of histone H3 biological consequences of different degrees of lysine Lys-4 methylation with euchromatin. At the individual gene methylation. level, methylation of this residue is usually (but not always) It should also be noted that there may be separate mech- correlated with active transcription. Several key observations anisms for using histone modifications to establish global vs. support the first point. Methylation of histone H3 at Lys-4 has gene-specific patterns of chromatin structure. At our current been correlated with transcriptionally active macronuclei of level of understanding, some histone modifications appear to Tetrahymena (7) and with euchromatic regions in fission yeast be associated with establishment of broad regions of chro- Schizosaccharomyces pombe (37). This general association also matin structure (e.g., heterochromatin vs. euchromatin), holds true in vertebrates (38, 39). For example, the highly whereas other histone modifications appear to be regulated transcribed ␤-globin locus of 10-d erythrocytes of chicken at the level of the individual gene or even specific regions of embryo contains dimethylation of H3 Lys-4 within the 30-kb a gene (e.g., promoter vs. coding region) and are involved ␤-globin locus, whereas the adjacent 15 kb of condensed, with regulating transcription of a specific euchromatic gene. inactive chromatin did not contain this modification (38). With regard to gene-specific regulation, deletion of the major C. Functional implications of methylation of individual histone H3 Lys-4 HMT Set1 in the budding yeast S. cerevisiae lysine residues of histones resulted in the repression of most genes (5059/6144 ORFs; 80%) as determined by microarray analysis (40). In addition, As discussed above, most histone modifications have been it was shown that dimethylation of H3 Lys-4 is associated primarily associated with either activation or repression of with the coding regions of active genes in S. cerevisiae (41). A transcription. However, for methylation at many lysine res- subsequent analysis using newly developed antibodies then idues, there are indications of its involvement in both acti- showed that it was the trimethylated Lys-4 that is highly 152 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription correlated with active genes, whereas dimethylated Lys-4 is iii. Recruitment of Set1 HMT: role of histone H2B monoubiq- a mark for both active and inactive euchromatin based on the uitylation. Another mechanism controlling Lys-4 methylation analysis of seven different genes in S. cerevisiae (42). These was elucidated by the discovery that monoubiquitylation of results have suggested that Lys-4 dimethylation is a mark of histone H2B at Lys-123 regulates dimethylation of histone H3 transcriptional permissiveness that functions to demarcate at Lys-4 and Lys-79 in S. cerevisiae (49–52) (Fig. 4). The E2 euchromatic from heterochromatic regions, whereas the tri- ubiquitin conjugating enzyme Rad6 is responsible for H2B methylation event at this same residue, which is restricted monoubiquitylation, and its deletion results in the elimina- primarily to the 5Ј end of genes, plays a direct transcriptional tion of global H2B monoubiquitylation and dimethylation of role. Unlike in S. cerevisiae, however, dimethylation of Lys-4 histone H3 at Lys-4 and Lys-79, but not Lys-36. In contrast, in metazoans is not found to occur broadly throughout gene mutation of histone H3 Lys-4 to Arg had no effect on H2B loci, but rather is found to track similarly with trimethylation monoubiquitylation, demonstrating a unidirectional regula- of Lys-4, which occurs predominately, but not exclusively, in tory path for controlling the global levels of H3 methylation. the promoter and 5Ј end of genes (43, 44). Thus, both the di- These studies provide a new paradigm whereby modifica- and trimethylated Lys-4 forms in multicellular organisms tions on one histone can regulate the outcome of modifica- appear to function in the activation process. tions on a different histone, hence the term “trans-histone” regulation. Bre1, an E3 ubiquitin ligase, is found to associate ii. Recruitment of Set1 HMT: roles of RNA polymerase II (pol with Rad6 and is responsible for the targeting of Rad6 to II) phosphorylation and PAF1 complex. How then is Set1 re- chromatin in S. cerevisiae. As expected, the deletion of Bre1 cruited to the 5Ј coding region of active genes and to eu- also leads to loss of monoubiquitylated H2B at Lys-123 and chromatin? So far, the mechanism does not appear to be global loss of Lys-4 and Lys-79 dimethylation (52, 53). directly dependent on transcriptional activators but instead A significant advance in our understanding of trans-tail depends on the RNA pol II elongation machinery, at least in histone regulation and Lys-4 methylation came from studies S. cerevisiae (45, 46). The elongation process of transcription showing that components of the Paf1 complex are also re- is regulated by phosphorylation of the C-terminal domain quired for global H2B Lys-123 monoubiquitylation and thus, (CTD) of RNA Pol II and by elongation factors such as the Lys-4/Lys79 methylation (54–56). Like Set1, Rad6 associates Paf1 complex. The CTD of RNA Pol II consists of a long series with the elongating polymerase via the Paf1 complex, and of heptapeptide repeats, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The Kin28 inactivation (i.e., the loss of CTD phosphorylation at phosphorylation status of the CTD correlates with the stages Ser-5) results in elimination of H2B monoubiquitylation (Fig. of RNA pol II in the transcription process. Ser-5 phosphor- 4). Thus, the association of Rad6 with Pol II appears to be ylation of the CTD is important for facilitating the transition essential for its catalytic activity (56). from transcription initiation to elongation and correlates Given that Rad6 and H2B ubiquitylation do not contribute with the promoter and the early phase of transcriptional directly to the recruitment of Set1, it is likely that H2B ubiq- elongation; Ser-2 phosphorylation of the CTD is associated uitylation functions to create an environment in chromatin with the late phase (i.e., downstream coding region) of tran- that is permissive for Set1 and Dot1 methylation (45). Be- scription elongation. In S. cerevisiae, Ser-5 is phosphorylated cause Rad6 and H2B monoubiquitylation track with elon- by the TFIIH-associated Kin28, whereas Ser-2 is phosphor- gating Pol II, it has been suggested that H2B monoubiqui- ylated by Ctk1 (Fig. 4) (47, 48). tylation may disrupt the nucleosomes surrounding Pol II, Several cooperative mechanisms are thought to contribute thereby making them accessible to the cotraveling HMTs to the recruitment of Set1 to 5Ј coding regions of actively including Set1. In addition, a new study reveals that pro- transcribed genes. First, Set1 specifically associates with Pol teasomal ATPases are recruited to ubiquitylated H2B and are II when the CTD is phosphorylated at Ser-5 but not at Ser-2 required for Lys-4 and Lys-79 methylation of H3 (57). These (45) (Fig. 4). In S. cerevisiae, deletion of Kin28 leads to sig- studies link proteasome function to the establishment of nificant loss of recruitment of Set1 to the 5Ј coding region in Lys-4 and Lys-79 methylation and suggest that chromatin the PYK1 gene; this suggests that the newly initiated poly- remodeling is required for some HMTs to recognize their merase acts as a signaling platform for the recruitment of sites of methylation in chromatin. Set1, resulting in Lys-4 trimethylation of the core promoter iv. H3 Lys-4 methylation in transcriptional activation by and early coding region (45). Second, components of the Paf1 nuclear receptors and other DNA-binding transcription factors. transcription elongation complex interact with Set1 and are Although Set1 is the only enzyme in yeast responsible for also required for recruitment of Set1 (46) (Figs. 2 and 4). Lys-4 methylation, this enzyme belongs to the trithorax Although the relationship between Ser-5 CTD phosphory- group of genes, and a number of homologs have been lation and Paf1-Set1 recruitment is unclear, it is possible that identified in other species (58, 59). In Drosophila, TRR, a the Paf1 elongation complex recruits Set1 in a manner that is trithorax-related SET domain protein that di- and trimethy- stabilized by the binding of Set1 to the Ser-5-phosphorylated lates histone H3 at Lys-4, can be recruited to promoters by CTD. Although these mechanisms have not been examined interacting with the ecdysone receptor, which is a DNA- in more complex eukaryotes, the observation that trimethy- binding transcription factor in the nuclear receptor family lation of Lys-4 occurs primarily in the promoter and 5Ј cod- (60). In Drosophila S2 cells, TRR is recruited to the pro- ing regions of active genes (43, 44) suggests that the recruit- moters of hedgehog and BR-C Z1 genes by the ecdysone ment of Set1 via the Paf1 complex and Pol II will most likely receptor in an ecdysone-dependent manner. Furthermore, be conserved. two different trr truncation mutants displayed significant Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 153 decreases in the mRNA level and the promoter-trimethy- functions in budding yeast may also be controlled by Lys-4 lation level of ecdysone-responsive genes. The human ho- methylation, directly or indirectly. molog of Drosophila trithorax, MLL/ALL1/HRX, is also 2. H3 Lys-79 methylation recruited to the promoter of Hox c8 gene (61, 62), suggesting that other types of DNA-binding transcription factors a. Activation. Dot1 is a unique HMT because it does not may recruit this HMT to promoters. Finally, Herr and contain a SET domain and methylates Lys-79 of histone H3, colleagues (63) identified the human Set1 homolog and which is located in the core rather than the tail of the nu- showed it to be involved in mammalian cell proliferation. cleosome (24, 73–75). The distribution of H3 Lys-79 methyl- Other Lys-4 HMTs such as Set7/9 have been identified (64, ation is similar to that of H3 Lys-4 methylation at both global 65), but their functions in chromatin are poorly defined. and gene-specific levels (Fig. 3). At the global level both Nevertheless, all of the Lys-4 HMTs appear to play a modifications are associated with euchromatin. In S. cerevi- significant role in gene activation. siae, H3 Lys-79 dimethylation is present in the euchromatin but not in the heterochromatic rDNA, telomere, and silent b. Role of H3 Lys-4 methylation in repression in budding mating type regions (76). In higher eukaryotes, active chro- yeast: direct or indirect effects? Although overwhelming ev- matin regions within the ␤-globin, Ig heavy chain, and TCR␤ idence associates histone H3 Lys-4 methylation with gene chain loci were enriched in H3 Lys-79 methylation in specific activation, there are some cases in which this modification hematopoietic cell lineages (76, 77). is implicated in gene repression, most notably in budding A recent model proposes that methylation of H3 Lys-79 yeast. In fact, Set1 was originally identified as a protein functions in budding yeast by inhibiting binding of Sir2/3 important in gene silencing in S. cerevisiae (36, 66, 67). In proteins, which deacetylate histones and help to establish budding yeast, three regions [mating type loci, telomeres, heterochromatin in silenced regions such as telomeres, and ribosomal DNA (rDNA)] display heterochromatin- rDNA, and mating type regions (Figs. 3 and 4). Deletion like behavior. Genes placed near or within these regions or overexpression of Dot1 results in mislocalization of become silenced in a manner similar to the position effect Sir2/3 from silenced regions (24, 73, 78), suggesting a variegation (PEV) phenomenon observed in Drosophila. complex role for H3 Lys-79 methylation in regulating Deletion of the SET1 gene in S. cerevisiae led to disruption Sir2/3 localization. It is not clear how H3 Lys-79 methyl- of silencing of an artificial reporter gene integrated in ation is restricted from heterochromatin and how this telomeres (66, 67), mating type loci and rDNA (36, 68). In modification regulates Sir2/3 localization. Di- and tri- addition, deletion of RAD6 or mutation of H2B Lys-123 to methylation of H3 Lys-4 inhibits binding of the nucleo- Arg caused defects in silencing of a URA3 reporter gene some remodeling and deacetylase (NuRD) corepressor in the telomeres (49, 69). Furthermore, deletion of SET1 or complex (Figs. 2 and 4) (which also contains histone mutation of Lys-4 to Arg caused an increase in expression deacetylases) and also inhibits H3 Lys-9 methylation by of an integrated reporter gene at the rDNA locus along Suv39h1 (65, 79), but whether methylation of H3 Lys-79 with loss of Lys-4 dimethylation in the same region (36, has similar consequences is not known. It is interesting to 68). Thus, it appears that Set1 plays a complex role in gene note that deletion of SET1 and DOT1 in S. cerevisiae syn- activation and repression in budding yeast, and more ergistically reduces the occupancy of Sir2 at the telomere, suggesting cooperative mechanisms for Set1 and Dot1 in work will be required to understand how it participates in establishing and maintaining euchromatin and hetero- both processes. However, another interpretation of the chromatin (54). above data is that the loss of Lys-4 methylation may affect Mechanisms for recruitment of Dot1 to euchromatin are gene silencing by an indirect mechanism (70). This was similar to Set1 at a global level. In budding yeast, H2B Lys- suggested by the fact that the silenced regions in budding 123 monoubiquitylation and components of the Paf1 com- yeast appear to be hypo-methylated at Lys-4 (41, 71). If the plex control both Lys-79 and Lys-4 methylation of histone H3 distribution of Lys-4 methylation (low in silenced regions, (Fig. 4) (46, 52). At a gene-specific level, dimethylated Lys-79 high in potentially active regions of chromatin) helps to is present at a similar level in the promoter and the coding restrict Sir silencing proteins to specific (silent) chromatin regions of active genes in S. cerevisiae (76). Dot1 occupancy, regions, then global loss of Lys-4 methylation could lead however, was more enriched in the coding regions than the to a redistribution of the Sir silencing proteins, thereby promoters (54). resulting in increased expression of genes in loci that are It is interesting to note that the steady-state level of H2B normally silenced. monoubiquitylation at Lys-123 is very low, around 5%, So far, evidence implicating H3 Lys-4 methylation in gene whereas the levels of H3 Lys-4 and Lys-79 methylation are silencing seems to be restricted to budding yeast. In fission much higher, 34 and 90%, respectively in S. cerevisiae (49). yeast, H3 Lys-4 dimethylation was not present in the mating The lower steady-state level of the ubiquitin modification type or the centromere regions, and in contrast to the silenc- may be due to the reversibility of this modification by deu- ing defect observed in budding yeast, deletion of Set1 did not biquitylating enzymes and suggests that the ubiquitin mod- lead to loss of silencing of a reporter gene integrated in those ification does not have to persist to maintain H3 Lys-4 and regions (72). The association of Lys-4 methylation with re- Lys-79 methylation (80–82). The higher levels of H3 Lys-4 pression in budding yeast may be explained by the fact that and Lys-79 methylation suggest that they may not be re- this organism lacks Lys-9 methylation (36). Thus, while Lys-4 versible or at least that they have a longer half-life; the ques- methylation is clearly associated with active genes, silencing tion of reversibility of lysine methylation is still unanswered. 154 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

In any case, histone H2B monoubiquitylation may play a regions of genes is responsible for repression of transcription, central role for establishing and/or maintaining euchroma- whereas coding region methylation at H3 Lys-36 could be tin, and thus understanding how the Bre1-Rad6 complex is associated with active transcription (83). How Set2 and H3 recruited and regulated will shed light on this process. Lys-36 methylation is directed toward specific promoters is not clear. Presumably, a different targeting mechanism is 3. H3 Lys-36 methylation responsible for the histone H3 Lys-36 methylation in the promoter region and coding regions. Alternatively, the a. Activation. The Lys-36 residue of histone H3 lies at the mono- and trimethyl forms of Lys-36, if they exist, may junction between the histone tail and core domains (25). function to regulate different aspects of the transcription Because of its unique location, methylation of this residue process than the dimethyl forms. Nevertheless, the dual roles could thus exert an effect by directly altering nucleosome of H3 Lys-36 methylation on transcription activation and structure or by promoting or inhibiting binding of chromatin repression may provide another example of the complexity remodeling proteins that recognize this mark. Currently, the of the histone code. role of H3 Lys-36 methylation in global euchromatin or heterochromatin formation is not clear. But, because methyl- 4. H3 Lys-9 methylation ation of H3 Lys36 does not occur at the telomeres and rDNA a. Repression regions of S. cerevisiae, it is likely that this modification is associated with euchromatin (83). i. Role of H3 Lys-9 methylation in PEV. The phenomenon of At the individual gene level, this modification is associated PEV in Drosophila provided a critical entry point for begin- with active genes, similar to H3 Lys-4 methylation (25, 83– ning to dissect the role of histone lysine methylation in het- 87). In S. cerevisiae, the histone H3 Lys-36 HMT, Set2, pref- erochromatin formation and maintenance (34, 35). PEV is a erentially binds to the Ser-2-phosphorylated vs. the unphos- gene-silencing event that results from chromosomal rear- phorylated CTD of RNA Pol II (83), suggesting a mechanism rangements such as inversion. As a consequence, a euchro- for Set2 recruitment to the coding regions of the transcribed matic gene is brought near the pericentric heterochromatin genes (Fig. 4). Furthermore, deletion of approximately 10 and becomes silenced due to a change in chromatin structure heptapeptide repeats of the CTD of RNA pol II resulted in a to heterochromatin. The fact that not all Drosophila cells in- significant global loss of histone H3 Lys-36 methylation, activate a euchromatic gene that has been newly juxtaposed while having no effect on the Lys-4 or Lys-79 methylation to pericentric heterochromatin suggests that spreading of level (83, 87). Deletion of individual components of the Ctk heterochromatin is a dynamic and regulated process. In sup- complex also led to complete loss of H3 Lys-36 methylation port of this, the genetic evidence for many other genes that at a global level, providing strong evidence that Ser-2 phos- serve as modifiers of PEV further supports that conclusion. phorylation controls H3 Lys-36 methylation by providing a Modifiers of PEV can enhance or suppress gene inactivation recruitment signal for Set2. As with Set1 recruitment, the of the repositioned euchromatic gene. Such modifier genes Paf1 complex also plays an important role in the recruitment were identified by selecting for random mutations that affect of Set2 (86). Thus, both the Paf1 complex and Ser-2 phos- PEV. Mutations in Su(var) genes, including Su(var)3–9 and phorylation of CTD work together to target Set2 to coding su(var)2–5/HP1, suppress PEV and thus reduce gene inac- regions of actively transcribed genes. tivation normally associated with juxtaposing a euchromatic In contrast to the positively correlated relationship be- gene with heterochromatin (89). This implies then that the tween H2B Lys-123 monoubiquitylation and H3 Lys-4 meth- wild-type Su(var) proteins contribute to gene inactivation by ylation, H2B monoubiquitylation appears to inhibit H3 PEV. Su(var)3–9 encodes a Lys-9 HMT that is homologous Lys-36 methylation. Mutation of H2B Lys-123 to Arg in S. to the mammalian Suv39h1 and Suv39h2 proteins (29). cerevisiae led to a dramatic increase in the dimethylation of Su(var)2–5/HP1 encodes the HP1 protein that is known to be H3 Lys-36 in the GAL1 promoter along with activated tran- involved in establishing heterochromatin (89, 90). HP1 con- scription (80). Set2 and H3 Lys-36 dimethylation occur on tains a chromodomain and a related chromoshadow domain. both the promoter and coding regions of several genes, al- A breakthrough in understanding of the mechanism of het- though the relative levels of Set2 and Lys-36 methylation are erochromatin formation resulted from the demonstration higher in the coding regions (83, 86). However, because not that the chromodomain of HP1 can specifically recognize all genes are methylated at H3 Lys-36, it appears that there methylated Lys-9 of histone H3 (91, 92), indicating that a is a gene selective targeting mechanism for Set2 (83). Lys-9 HMT and HP1 are mechanistically linked and act together to establish heterochromatin. b. Repression. Similar to Set1, Set2 also was originally implicated in transcriptional repression in budding yeast (25, ii. Different HMTs for H3 Lys-9 methylation in euchromatin 88). Tethering of Set2 to the promoter of a reporter gene by and heterochromatin. Different degrees of Lys-9 methylation fusing Set2 to a DNA-binding domain led to repression of correlate with distinct chromatin regions, and it appears reporter gene expression, and the repression was relieved that the function and regulation of Lys-9 methylation is partially by mutations in the SET domain (25). Furthermore, different in heterochromatin vs. euchromatin (93–95). In Set2 is required for maintaining low basal expression of the constitutive pericentric heterochromatin, Suv39h1/2 me- GAL4 gene in S. cerevisiae (88). Deletion of Set2, mutation of diates trimethylation of H3 Lys-9, whereas in euchroma- the catalytic site of Set2, or mutation of histone H3 Lys-36 to tin, the HMT G9a mediates dimethylation of H3 Lys-9 in Arg led to elevation of the basal level of GAL4 gene expres- vivo (93, 94). It is interesting to note that in vitro, both sion. It is possible that methylation of H3 Lys-36 in promoter Suv39h1 and G9a can convert a histone H3 peptide with Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 155 dimethylated Lys-9 to the trimethyl form, whereas in vivo methylation of the centromeric region. These observations they display very different characteristics. In Suv39h1/2 suggest that shRNA in heterochromatic regions helps to re- double-null mouse embryo fibroblasts, trimethylation of cruit Clr4, which establishes Lys-9 methylation that then H3 Lys-9 is abolished, whereas mono- and dimethylation recruits Swi6. Consistent with the knowledge that histone were not significantly affected. In contrast, in G9a null deacetylases facilitate the initial stages of assembly of het- mouse embryo fibroblasts, there was a complete disap- erochromatin, Clr3, which deacetylates H3 Lys-14, was par- pearance of dimethylation of H3 Lys-9, a significant de- tially required for the H3 Lys-9 methylation and recruitment crease in monomethylation, and no change at the trim- of Swi6 to the centromere (98). Evidence that heterochro- ethylation level. In addition, trimethylation of Lys-9 is a matic shRNA can directly recruit Suv39h1 is currently lack- property of pericentric heterochromatin, whereas dim- ing. However, two different mutations in the chromodomain ethylation is dispersed throughout the euchromatin, sug- of Clr4 lead to loss of H3 Lys-9 methylation and Swi6 local- gesting that mono-, di-, and trimethylation at Lys-9 are ization in vivo, whereas in vitro they do not affect Clr4 meth- differentially regulated and can exert different functional yltransferase activity (98). One possible explanation for these outcomes, as with Lys-4 methylation. Thus, Suv39h1/2 is findings is that the chromodomain of Suv39h1 may be im- the major Lys-9 trimethylase in pericentric heterochroma- portant in the recognition of shRNA. The chromodomains of tin, and G9a is the major H3 Lys-9 dimethylase in two other proteins (histone acetyltransferase MOF and male- euchromatin. specific lethal protein MSL3) have been shown to have RNA These two enzymes also display different chromosomal binding activity (102, 103). localization patterns, implying different modes of recruit- Once HP1/Swi6 has been recruited to initiate heterochro- ment. Whereas Suv39h1 is associated with pericentric het- matin formation, it also initiates the spreading of hetero- erochromatin and colocalizes with HP1␣/␤, G9a is associ- ␣ ␤ chromatin by self-association with other HP1 molecules and ated with euchromatin and does not colocalize with HP1 / by using its chromoshadow domain to recruit additional (96, 97). Whereas Suv39h1 is recruited to heterochromatin Suv39h1, which further catalyzes Lys-9 methylation to attract through its N-terminal chromodomain (98), G9a lacks a chro- more HP1, and so forth (37). How these events lead to gene modomain; instead, G9a contains an ankyrin-repeat domain, silencing and how the spreading of heterochromatin is reg- which is also implicated in protein-protein interactions (96) ulated are also important questions for future investigation. and thus may play an important role in G9a targeting. In The role RNA plays in the formation of centromeric hetero- addition, whereas Suv39h1 is inhibited by H3 Lys-4 di- chromatin in S. pombe also seems likely to be true in higher methylation in vitro, G9a is not (65). It is also interesting to eukaryotes. In permeabilized human cells, dimethylation of note that some H3 Lys-4 HMTs, such as Set7/Set9 and MLL/ histone H3 at Lys-9 and recruitment of HP1 in pericentric ALL1, also are not inhibited by H3 Lys-9 dimethylation in heterochromatin were abolished by RNase treatment (95). vitro (62, 65). Because this implies that both modifications can Furthermore, addition of total or nuclear RNA restored the coexist on the same histone tail, it is currently unclear how methylation and HP1 localization pattern, suggesting that the antagonism between H3 Lys-4 methylation (which is generally thought to be associated with active or potentially RNA is an important component of the pericentric hetero- active chromatin) and H3 Lys-9 methylation (which is gen- chromatin. The identity of the RNA has not been determined erally thought to be associated with inactive chromatin or but is presumably similar to heterochromatic shRNA ob- heterochromatin) is resolved. served in S. pombe. iii. Recruitment of Suv39h1 HMT by short heterochromatic iv. Recruitment of H3 Lys-9 HMTs to euchromatic promoters RNAs (shRNAs) in formation of pericentric heterochromatin. by sequence-specific DNA-binding transcriptional repressor pro- How does the Suv39h1 HMT recognize which regions of teins. Although methylation by Suv39h1 has been primarily cellular chromatin to methylate? Genetic evidence indicated associated with the establishment and maintenance of het- that Suv39h1 lies upstream of HP1 action (91, 98), but how erochromatin, there are examples of its involvement in re- Suv39h1 is targeted to assemble heterochromatin was un- pressing genes in mammalian euchromatin. The retinoblas- clear until a surprising discovery implicated repetitive DNA toma (Rb) protein is part of a corepressor complex that binds elements and RNA interference (RNAi) machinery in re- to the E2F transcription factors to repress transcription of cruiting Clr4 (the S. pombe equivalent of Suv39h1) to the genes required for cell cycle progression. Phosphorylation of centromeric heterochromatic region of S. pombe (99–101). Rb at a particular stage of the cell cycle causes Rb to dissociate Centromeric repeats are transcribed bidirectionally to pro- from E2F and thus allows cell cycle progression. The Rb duce noncoding double-stranded RNA, which is processed corepressor complex contains histone deacetylases and also to small interfering RNA (siRNA, also called short hetero- Suv39h1. Suv39h1 methylation of Lys-9 of histone H3 results chromatic RNA or shRNA) by the RNAi machinery (99). in recruitment of HP1 to the cyclin E gene promoter and Deletion of any of the three components of RNAi machinery represses its transcription (Fig. 2) (104, 105). Similarly, [RNAseIII helicase dicer (dic1), RNA-dependent RNA poly- KRAB-ZFP, which is a DNA sequence-specific transcrip- merase (rdp1), and Argonaute (ago1)] caused inappropriate tional repressor protein, recruits the KAP1 corepressor that activation of a reporter gene integrated within centromeric brings the H3 Lys-9 HMT SETDB1/ESET to promoters of heterochromatin. In addition, loss of centromeric localization specific genes and results in transcriptional silencing due to of Swi6 (the S. pombe equivalent of HP1) and H3 Lys-9- histone H3 Lys-9 methylation and HP1 deposition (106). dimethylation were observed, along with increased H3 Lys-4 Similarly, G9a is specifically targeted to the promoter of the 156 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription interferon ␤ gene by the DNA-binding PRDI-BF1 repressor 5. H3 Lys-27 methylation protein (107). a. Repression. Methylation of histone H3 at lysine 27 dis- ESET, a SET domain protein that associates with an ets- plays two functional similarities to that of lysine 9. First, related-gene transcription factor, is regulated by mAM, a different degrees of methylation of both marks have different murine activating transcription factor-associated modulator, distributions in the chromatin. Monomethylation of Lys-27 is which was biochemically identified as a protein tightly as- found in pericentric heterochromatin along with trimethy- sociated with ESET (108, 109). mAM increased the enzymatic lation of Lys-9 (93, 94) (Fig. 3). On the other hand, trimethy- activity of ESET and also changed its substrate specificity so lation of Lys-27 is characteristic of facultative heterochro- that it produced trimethyllysine instead of only dimethyl- matin of the inactive X chromosome during the initial stage lysine at Lys-9 of histone H3. Furthermore, in transcription of X inactivation (114, 115). The inactive X chromosome also assays using chromatin templates, trimethylation of H3 displays dimethylated but not trimethylated Lys-9 (114, 116– Lys-9 at the promoter region by mAM/ESET leads to tran- 118). There are specific functional consequences associated scriptional repression, whereas dimethylation of H3 Lys-9 by with the number of methyl groups on a specific histone lysine ESET alone has only a modest effect (109). These findings residue. HP1 colocalizes with Lys-9 trimethylation in peri- suggest possible mechanisms for regulating the number of centric heterochromatin, but not with dimethylated H3 Lys-9 methyl groups on a specific histone lysine residue by regu- in the inactive X chromosome (95, 116, 117). In addition, lating cellular levels and promoter localization of specific Suv39h double-null mouse embryonic fibroblasts still main- HMTs, or availability of HMT-associated proteins that mod- tain Lys-9 dimethylation of inactive X, indicating that a dif- ulate HMT activity. It also suggests putative mechanisms for ferent HMT is responsible for the methylation of the inactive physiological regulation of the activity and substrate spec- X. EZH2, a mammalian homolog of Drosophila enhancer of ificity of at least some HMTs by intracellular signaling or cell zeste [E(z)] is the HMT that mediates methylation of H3 context. Lys-27 of inactive X chromosome; this enzyme also methy- b. Activation: possible roles for H3 Lys-9 methylation in specific lates H3 Lys-9 in vitro, but whether it does so on the inactive cases of transcriptional activation? Although most H3 Lys-9 X chromosome in vivo is not clear (119–122). E(z) belongs to methylation appears to be involved in heterochromatin for- the polycomb (Pc) group of proteins that are involved in mation and gene repression, a few observations hint at pos- long-term silencing of Hox genes. The Pc group contains two sible selective involvement in gene-specific transcriptional major complexes, ESC-E(z) and Pc repressive complex-1 activation. Ash1, a member of the trithorax group in Dro- (PRC1). sophila, is an unusual HMT because it can methylate histone The second similarity is that both modifications create H3 at Lys-4 and Lys-9 and histone H4 at Lys-20 in vitro (110); binding sites for the recruitment of specific effector proteins however, in vivo Ash1 is responsible for the majority of H3 that contain chromodomains. Although Suv39h1-mediated Lys-4 methylation but not for the majority of H3 Lys-9 and trimethylation of histone H3 Lys-9 leads to recruitment of H4 Lys-20 methylation (111). It is still possible, though, that HP1 in mammals, ESC-E(z) complex mediated methylation Ash1 can mediate methylation of H3 Lys-9 and H4 Lys-20 at of histone H3 Lys-27 creates a specific binding site for the specific loci in vivo. Chromatin immunoprecipitation exper- recruitment of the PRC1 via Pc protein in Drosophila (123, 124) iments demonstrated that dimethylation of Lys-4 and Lys-9 (Fig. 2). The chromodomain of Pc protein specifically recog- of histone H3 and Lys-20 of histone H4 is linked with tran- nizes trimethylated Lys-27 of histone H3. scriptional activation of Ash1 target genes, both an inte- Two different mechanisms exist for recruiting H3 Lys-27 grated reporter gene and the endogenous Ultrabithorax (Ubx) HMTs to their targets. At the global level, EED-EZH2, the gene (110). HP1, on the other hand, was not present on the human ESC-E(z) complex, is recruited to inactive X chro- promoters of the integrated reporter gene or the endogenous mosome via Xist RNA to trimethylate histone H3 at Lys-27; Ubx when they were active. These observations demonstrate this is similar to the mechanism by which centromeric that dimethylation of H3 Lys-9 can be associated with acti- shRNA recruits Clr4 (equivalent of human Suv39h1) to het- vated transcription in the presence of H3 Lys-4 and H4 erochromatin in fission yeast (Fig. 2) (114, 115). Interestingly, Lys-20 methylation, suggesting the possible importance of recruitment of EED-EZH2 and trimethylation of H3 Lys-27 context specificity in dimethylated Lys-9 for its biological are transient, occurring only during the initial stage of X consequences. inactivation. At the individual gene level, Drosophila ESC- Like H3 Lys-9 methylation, HP1 is usually associated with E(z) complex is targeted to Pc response elements via many inactive chromatin. However, evidence in Drosophila indi- DNA binding proteins such as GAGA factor, pleiohomeotic cates that HP1 is associated with active genes in at least a few (Pho), and Zeste (119, 125–128). For example, E(z), Pc and dimethylation of H3 Lys-27 are all targeted to the region of specific cases. In larval salivary gland polytene chromo- the Pc response element of the Drosophila Ubx gene to repress somes, HP1 is associated with ecdysone and heat-shock in- transcription (122). Furthermore, mutations in the SET do- duced puffs of euchromatin, in addition to its localization at main of E(z) led to loss of repression of Ubx in wing imaginal the heterochromatic regions (112, 113). In chromatin immu- discs in vivo, demonstrating that HMT activity is important noprecipitation analysis of cultured Drosophila S2 cells, heat- for Pc group-mediated gene silencing (120). shock treatment resulted in recruitment of HP1 to the coding region of the Hsp70 gene, but not the promoter. Whether H3 6. H4 Lys-20 methylation. Methylation of histone H4 at lysine Lys-9 methylation is responsible for the recruitment of HP1 20 is mediated by the PR-Set7/Set8 HMT (26, 27). In Dro- in these cases remains to be determined. sophila polytene chromosomes, this modification is associ- Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 157 ated with the chromocenter and euchromatic arms. Staining scription factor that binds TATA boxes and thus sets the in the euchromatin does not significantly colocalize with transcription start site for TATA box-containing genes and dimethylated Lys-4 of histone H3, suggesting a role in the helps to assemble the other basal transcription factors and silent domains of euchromatin. RNA pol II on the promoter. Lysine methylation of TAF10 increases its affinity for RNA pol II. Furthermore, loss of D. Consequences of histone lysine methylation SET9 methyltransferase activity or mutation of the TAF10 methylation site caused a reduction in transcriptional acti- It is clear that histones, especially the N-terminal tails, vation of specific transient reporter genes as well as specific provide a platform for convergence and integration of signals endogenous genes (130). Because very few proteins have into a distinct biological outcome, in this case regulation of actually been examined for lysine methylation at this point, transcription. How does a methyl mark then lead to a distinct this report is presumably one of the first of what will be many biological outcome? There are three possible mechanisms reports in the next few years of lysine methylation of non- that may not be mutually exclusive. First, methylation of histone proteins. lysine residues could inhibit binding of proteins (or other nucleosomes) to histone tails or inhibit binding or activities of enzymes that make additional modifications of histone III. Arginine Methylation tails or other proteins. Second, methylation could create a A. Enzymes binding site for the recruitment of specific proteins involved in chromatin remodeling or enhance activities of certain en- 1. The protein arginine methyltransferase (PRMT) family. Meth- zymes to make additional histone modifications. Third, hi- ylation of arginine residues is a common posttranslational stone methylation in strategic regions of the nucleosome modification in eukaryotes. Two types of PRMTs transfer the could affect nucleosome conformation and thus its interac- methyl group from AdoMet to the guanidino group of ar- tion with other proteins or nucleosomes. A few specific ex- ginines in protein substrates. Type I PRMT enzymes form amples illustrating these mechanisms are summarized here monomethylarginine and asymmetric dimethylarginine (Figs. 2 and 4). H3 Lys-4 dimethylation, for example, inhibits products. Type II PRMT enzymes catalyze the formation of binding of mammalian NuRD corepressor complex, which monomethylarginine and symmetric dimethylarginine (131, contains histone deacetylase and an ATP-dependent chro- 132) (Fig. 1C). PRMTs may be universal to all eukaryotes, matin remodeling activity (79). In contrast, Lys-4 methyl- because one or more representatives are found in fungi, ation enhances mammalian p300 HAT activity and stimu- higher plants, invertebrates, and vertebrate animals (133). lates the recruitment of coactivator Isw1p ATPases in Seven mammalian PRMT genes have been identified: budding yeast (64, 65, 79, 129), although the interaction is PRMT1, PRMT2, PRMT3, CARM1/PRMT4, JBP1/PRMT5, apparently not direct and the domain/protein that can rec- PRMT6, and PRMT7 (Fig. 5); but the yeast S. cerevisiae has ognize methylated H3 Lys-4 is still not clear. H3 Lys-4 meth- only one member, Hmt1/Rmt1. PRMT5 is the only example ylation, in addition, inhibits enzymatic activity of the H3 of a type II enzyme, whereas the other PRMTs (except Lys-9 HMT Suv39h1 in mammals by serving as a poor sub- PRMT7) are all type I enzymes. PRMT 7 makes only mono- strate (65). H3 Lys-9 and Lys-27 methylation, on the other methylarginine and contains two methyltransferase domains hand, creates a high affinity binding site for the chromodo- in a single polypeptide chain (134) and thus may represent mains of Drosophila HP1 and Pc, respectively (123, 124). Al- a third class of PRMT. though Lys-9 and Lys-27 of histone H3 are both embedded The means by which the various PRMTs were discovered in the same local sequence context, Ala-Arg-Lys-Ser, HP1 suggests diverse roles for these enzymes in intracellular sig- and Pc can discriminate between the two methylated lysines naling. The discovery of protein arginine methyltransferase by reading the residues preceding the Ala-Arg-Lys-Ser se- activity (135, 136) preceded the isolation of the first cDNA quence. HP1 primarily reads residues nϪ1tonϪ3, where “n” clones for yeast Hmt1/Rmt1 (137, 138) and for mammalian is the methylated lysine. In contrast, Pc reads residues nϪ4 PRMT1 (139) by almost three decades. The PRMT1 gene was through nϪ7, contributing to differential protein recogni- discovered through interaction of PRMT1 with TIS21 and tion. As a consequence, Pc binds 25 times better to trimethy- BTG1, which are immediate-early proteins in the response to lated Lys-27 than to trimethylated Lys-9. HP1, on the other mitogens (139). PRMT1 was also found to associate with the hand, binds 13 times better to trimethylated Lys-9 than to intracytoplasmic domain of the interferon-␣/␤ receptor Lys-27. Clearly, the identification of additional proteins that (140). PRMT2, for which enzymatic activity has not yet been recognize specific sites of histone methylation will extend detected, was identified in expressed sequence tag databases our understanding of the histone code and how it regulates by its sequence similarity with PRMT1 (141). PRMT3 was transcription. found because of its binding to PRMT1 (142). CARM1 (coactivator-associated arginine methyltransferase-1) was E. Lysine methylation of a basal transcription factor identified as an interacting protein for the p160 transcriptional coactivator, glucocorticoid receptor-interacting pro- SET9, which was originally characterized as a H3 Lys-4 tein-1 (GRIP1) (6). JBP1 (Janus kinase-binding protein-1, also

HMT, was recently shown to methylate TAF10 (TAFII30), a referred to as PRMT5) was found through its interaction with subunit of the TFIID complex (130). TFIID, which consists of Janus kinase Jak2 (143, 144). PRMT6 was also identified be- the TATA box binding protein TBP and about 10 TBP asso- cause of PRMT sequence homology (145). Further analysis of ciated factors or TAFs of varying sizes, is the basal tran- protein interaction partners and protein substrates for these 158 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

FIG. 5. The PRMT family of proteins. The catalytic core regions common to all PRMTs consist of a highly conserved AdoMet binding region formed by a Rossman fold and two ␣ helices (orange), and a less conserved ␤-barrel structure (yellow) that folds against the AdoMet binding region to form the protein substrate binding cleft. Some family members contain additional recognizable motifs such as zinc fingers and SH3 domains. PRMT7 contains two catalytic domains within the same polypeptide chain; the two halves of the protein are aligned separately in the figure (PRMT7 N-terminal and PRMT7 C-terminal). enzymes will continue to be critical for elucidating their sisting of a typical Rossman fold and two ␣-helices, is the physiological roles. AdoMet-binding domain, the most highly conserved region among PRMTs and also partially conserved in other types of 2. Structure, catalytic mechanism, and substrate specificity AdoMet-dependent methyltransferases (150). The C-termi- a. Protein substrate specificity. PRMT proteins vary in length nal half of the core forms a barrel-like structure, unique to the from 348 (S. cerevisiae Rmt1) to 637 (PRMT5) amino acids; PRMT family, which folds against the N-terminal AdoMet- PRMT7, with duplicate methyltransferase domains, has 692 binding region. The resulting cleft provides a protein sub- amino acids. The methyltransferase activity resides in a strate binding site and the site of catalysis. The three-dimen- highly conserved core region of approximately 310 amino sional structure analysis also reveals a dimerization interface acids. In addition to the conserved methyltransferase do- that is essential for the enzymatic activity. Indeed, dimer main, each PRMT member has a unique N-terminal region formation encloses the active sites into a hole between the that varies widely in length, and CARM1 also has a unique two monomers. Mutation of dimer contact residues of Hmt1 C terminus (Fig. 5). Despite the high degree of conservation (149) or deletion of the PRMT1 dimerization arm (133) elim- within the methyltransferase domain, there is relatively little inates the enzymatic activity. The dimerization may contrib- overlap in the protein substrate specificities of the seven ute to the formation of dimethylarginine by facilitating trans- PRMTs (11, 145). The mechanism of specific substrate rec- fer of the monomethylated substrate from the active site of ognition is still unclear, and in fact based on the known one monomer directly into the active site of the second mono- substrates, few clear consensus recognition sequences have mer, adding the second methyl group without dissociation emerged. For example, PRMT1 methylates many substrates of the protein substrate from the dimeric enzyme. Some in regions containing Arg-Gly-Gly repeats (146), but not all PRMTs also have the ability to form larger homomeric ag- PRMT substrates have such sequences (147). CARM1 also gregates. Hmt1 (149) and PRMT1 (142) form hexamers in appears to have more than one type of sequence immediately solution, and PRMT5 also forms homooligomers of more surrounding the methylation sites of its substrates (148). than two subunits (144). The role of the multimerization is Although the crystal structures do not fully explain substrate not clear, although a PRMT5 multimer complex shows a high specificity, they may provide some clues about this mystery enzymatic activity, suggesting that the formation of homo- and also provide insight to the general mechanism of catal- oligomers is important for the efficient catalytic activity of ysis by these enzymes. Structures of the conserved core re- PRMT5 (144). The residues implicated in AdoMet binding, gion have been published for Hmt1 (149), PRMT3 (150), and in catalysis, in intramolecular contacts between the AdoMet- PRMT1 (133), in some cases in complex with S-adenosylho- binding region and the barrel-like domain, and in the mocysteine (AdoHcy; the product remaining after a methyl putative dimer interface are conserved across the PRMT group is extracted from AdoMet) and/or some substrate family, suggesting a common fold and catalytic mechanism peptides. On the surface of PRMT1 is a long, meandering but (149–150). possibly continuous groove which provides multiple sites c. Functions of unique N- and C-terminal regions of PRMTs. that can bind substrate peptides. Thus, it is conceivable that Whereas the functions of the unique N-terminal and C- these multiple sites could accommodate several different terminal regions of PRMTs are still not clear, deletion anal- types of peptide substrates and thus could explain the ap- yses have provided some clues to their functions. Deletion of parent ability of a single enzyme to recognize multiple con- the N terminus of Hmt1 impairs the stability of the hexamer sensus sequences, which could be at varying distances from and decreases the methyltransferase activity, suggesting that the actual site of methylation. multimer formation contributes to enzymatic activity (149). b. Structure and catalysis. In all three published structures, Similarly, an interaction between the N and C termini of the core region consists of two domains folded together into PRMT5 appears to be involved in its homomeric complex an integral structure. The N-terminal half of the core, conformation (144). Deletion of the N-terminal part of PRMT3, Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 159 which contains a zinc-finger motif, impairs its enzymatic signaling pathways. Many of these coactivators catalyze a activity and possibly alters protein substrate specificity, sug- variety of posttranslational protein modifications, and in fact gesting a role in protein substrate recognition (142, 151). The a dauntingly large number and variety of posttranslational unique C terminus of CARM1, which contains a strong au- modifications of histones and nonhistone proteins have been tonomous transcriptional activation activity, is required implicated in transcriptional regulation (14, 153). Studies along with the methyltransferase activity for the transcrip- with the nuclear receptor family of hormone-regulated tran- tional coactivator function of CARM1 (see Section III.B), and scriptional activator proteins (154, 155) have played a central thus may interact with other components of the transcription role in elucidating the role of coactivators in general and, in machinery that participate in the process of transcriptional particular, protein arginine methylation in transcriptional activation (152). activation. Binding of the appropriate hormone to nuclear receptors causes a conformational change that facilitates binding of many coactivators and thus presumably allows B. Effects of arginine methylation on protein function the DNA-bound receptor to recruit key coactivators to the For the most part, the specific changes in protein function promoter. Of the many coactivators that have been impli- resulting from protein arginine methylation have yet to be cated in nuclear receptor function (156, 157), only a few have determined. A few specific cases where such information is been functionally well characterized (Fig. 6). For example, available will be discussed below. More extensive previous the SWI/SNF complex possesses ATP-dependent chromatin studies on the functional effects of other protein modifica- remodeling activity; histone acetyltransferases such as tions, such as phosphorylation, can provide models to guide CREB-binding protein (CBP), p300, and p300/CBP-associ- our thinking about the functional ramifications and potential ated factor (pCAF) contribute to chromatin remodeling physiological roles of protein methylation and can suggest through a different mechanism; the TRAP/DRIP/mediator experimental strategies to test such ideas. While methylation complex associates with and is apparently involved in the should not affect the overall charge of an arginine residue, it recruitment and activation of RNA pol II. Coactivators of the can be expected to add bulk and hydrophobicity that can p160 family (SRC-1, GRIP1/TIF2, and pCIP/ACTR/RAC3/ promote or inhibit intra- or intermolecular interactions (with AIB1/TRAM1) bind only to the hormone activated form of proteins or other types of molecules) (Fig. 1C). Such altered the nuclear receptors and recruit additional coactivators, interactions may change the shape and thus the function or called secondary coactivators, including the protein/histone stability of the methylated protein, or may serve to facilitate acetyltransferases CBP and p300 (158, 159) and the protein/ or interfere with intermolecular (e.g., protein-protein or histone arginine methyltransferases CARM1 and PRMT1 (6, protein-RNA) interactions or enzymatic activities that play important roles in specific signaling pathways.

1. Implication of arginine methylation in transcriptional regulation. The arginine methyltransferases modify proteins that function at many different steps in cellular regulation, including cytoplasmic and nuclear signal transduction pathways, nuclear-cytoplasmic shuttling, transcriptional activation, and multiple posttranscriptional steps in gene expression. This section will focus on methylation of histones and nonhistone proteins involved in transcriptional regulation, followed by brief examples of protein methylation that may regulate posttranscriptional steps of gene expression. For perspective, subsequent sections will briefly discuss evidence implicating arginine-specific protein methylation in some cytoplasmic signaling pathways. FIG. 6. Role of PRMTs in transcriptional activation by nuclear re- a. Histone methylation and chromatin remodeling ceptors. Hormone-activated nuclear receptors (NR) bind to a specific hormone-responsive enhancer element (HRE) sequence in DNA and i. PRMTs as transcriptional coactivators for nuclear receptors. recruit various coactivator complexes, which help to remodel chromatin structure and recruit and activate RNA pol II complex and its Activation of transcription of a specific protein-encoding associated basal transcription machinery (including basal transcrip- gene generally involves binding of one or more transcription factors TFIIB and TBP; TBP binds to TATA boxes in the basal tional activator proteins to specific enhancer elements asso- promoter region). The SWI/SNF coactivator complex carries out ATP- ciated with the gene. The DNA-bound transcriptional acti- dependent chromatin remodeling. The p160 coactivator complex is anchored to the nuclear receptors through the p160 protein (GRIP1 vator protein recruits a variety of coactivator proteins that or SRC-1) and also contains histone acetyltransferases (CBP or p300 remodel chromatin in the promoter region into a more open and pCAF) and histone-methylating PRMTs (PRMT1 and CARM1). conformation and recruit and activate RNA pol II and the rest CARM1 has also been found to associate with the SWI/SNF complex of the basal transcription machinery. The large number of and thus may coordinate the activities of these two complexes. The coactivators that are apparently involved suggests that the TRAP/DRIP (also called mediator) complex helps to recruit and activate RNA pol II by association with the CTD of RNA pol II. Double- processes of chromatin remodeling and transcriptional acti- headed black arrows, recruitment of coactivators; single-headed black vation are exceedingly complex and subject to regulation at arrows, recruitment of basal transcription machinery by coactivators; many different steps and from many different interacting red arrows, enzymatic modifications of histones and other proteins. 160 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

160). The discovery of CARM1 as a histone methyltransferase efficiently than hyperacetylated histones H3 and H4. In this and a secondary coactivator for nuclear receptors (6) con- case, PRMT5 is recruited to c-Myc target genes with the other stituted the first implication of protein methylation in tran- components of the complex and appears to be involved in scriptional activation. CARM1 methylates histone H3 at gene repression (174). Arg-2, -17, and -26 (161) (Fig. 1A) and enhances transcrip- Thus, arginine-specific histone methylation is a part of the tional activation by nuclear receptors in transient transfec- transcriptional activation process and occurs in cooperation tion assays (6); both the methyltransferase activity and the with other histone modifications. Proof that these histone association with p160 coactivators are essential for CARM1 methylation events actually play important roles in tran- coactivator function with nuclear receptors (162, 163). More- scriptional activation has been provided recently from in over, in chromatin immunoprecipitation assays, CARM1 it- vitro transcription experiments using recombinant chromatin self and methylation of histone H3 at Arg-17 were associated templates (172). The transcriptional activator p53 binds di- with the hormone-inducible promoters of stably integrated rectly to p300, CARM1, and PRMT1 and recruits them to the reporter genes and endogenous (i.e., native) genes in a target promoter, where they make the appropriate histone hormone-dependent manner (164, 165). Thus, recruitment of modifications in an ordered fashion (PRMT1, p300, CARM1; CARM1 and arginine methylation of histone H3 are integral see above) and cooperate synergistically as coactivators. parts of the transcriptional activation process. Moreover, reconstitution of the chromatin template with hi- PRMT1 has also been confirmed as an arginine-specific stones containing mutations that prevent acetylation or histone methyltransferase; PRMT1 methylates histone H4 at methylation by one of these enzymes abolishes the ability of Arg-3 in vitro and in vivo (6, 166, 167) (Fig. 1A). Thus, it is not that enzyme (but not the other two enzymes) to enhance surprising that in transient transfection experiments, p53-mediated transcription. This work confirms that argi- PRMT1, which also interacts with the p160 cofactors, has nine methylation of histones not only occurs, but is important been shown to enhance transcriptional activation by nuclear for transcriptional activation. receptors in a manner that requires PRMT1 enzyme activity iii. PRMTs as coactivators for diverse types of transcriptional (160, 167). Chromatin immunoprecipitation also showed activator proteins. Although much of the evidence for the PRMT1 recruitment to a hormone-activated promoter (168). involvement of protein arginine methylation in transcrip- PRMT2 (169) and PRMT3 (S. S. Koh, C. Teyssier, H. Li, and tional regulation has come from studies involving nuclear M. R. Stallcup, unpublished observations) have also been receptors, recent findings indicate that CARM1 and PRMT1 reported as transcriptional coactivators for nuclear receptors, interact functionally with other classes of transcriptional ac- although no enzymatic activity for PRMT2 has yet been dem- tivators, such as LEF-1/TCF4 (175), p53 (172), and YY1 (176). onstrated, and no target for methylation by PRMT3 has yet Thus, protein arginine methylation is likely to be involved in been identified in connection with its coactivator activity. chromatin remodeling and transcriptional regulation by a ii. Functional interactions of histone acetylation and histone wide variety of DNA-binding transcription factors. arginine methylation. The fact that arginine methylation of iv. Possible consequences of histone arginine methylation. The histones occurs in the N-terminal tails among the sites for molecular mechanism(s) by which arginine methylation of lysine methylation, lysine acetylation, and serine phosphor- histones contributes to chromatin remodeling and transcrip- ylation (Fig. 1A) strongly suggests functional relationships tional activation are not known. The histone tails are external among histone modifications (1, 12, 170, 171). In fact, mul- to the nucleosome structure and therefore accessible not only tiple coactivators with histone modifying activities were for covalent modifications, but also for additional intermo- found to cooperate synergistically in transient transfection lecular interactions, e.g., with other nucleosomes to accom- assays: CARM1 (arginine methylation of histone H3) coop- plish chromatin compaction or with other proteins that par- erated with PRMT1 (arginine methylation of histone H4) ticipate in modulation of chromatin structure. The sequential (160); CARM1 (but not PRMT1, PRMT2, or PRMT3) coop- and interdependent histone tail modifications are clearly one erated with p300, CBP, and pCAF (acetylation of histones H3 example of this but must also lead to additional actions that and H4) (163). Both the PRMTs and p300/CBP are apparently result in chromatin remodeling and transcriptional activa- recruited as secondary coactivators for nuclear receptors by tion. Other possible downstream effects of arginine methyl- interaction with a p160 protein. One (but by no means the ation of histones include disruption of nucleosome stability only) possible explanation for the cooperative coactivator or internucleosomal interactions; disruption of binding by functions of these histone-modifying enzymes is that some of proteins that contribute to chromatin compaction or tran- the histone modifications may be facilitated by the prior scriptional repression; or creation of binding sites for pro- occurrence of the others. Indeed, methylation of free histone teins that promote a more open chromatin conformation or H4 by PRMT1 stimulated the subsequent acetylation of this contribute in some other manner to transcriptional activa- histone by p300 (167) and acetylation of nucleosomes in tion. It will be of a great interest to determine the proteins that p53-dependent transcription in vitro (172). Similarly, prior bind differentially to arginine-methylated vs. unmethylated acetylation by p300 enhanced CARM1 binding and enzy- histones and vice versa. matic activity on histone H3 tail peptides (173) and on as- sembled chromatin templates (172). On the contrary, PRMT5, v. Coordination of chromatin remodeling by PRMTs and ATP- when associated with the corepressor complex mSin3/his- dependent enzyme complexes. Another recent study suggests tone deacetylase 2 and Brg1 (the hSWI/SNF ATPase subunit) that histone methylation by CARM1 is coordinated with could methylate hypoacetylated histones H3 and H4 more ATP-dependent chromatin remodeling. CARM1 was found Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 161 as a component of a SWI/SNF-like complex (177). CARM1 tabolism including splicing, nuclear export, and stability. In physically interacts with Brg1 and stimulates its ATPase some cases discussed below, methylation effects on specific activity. Furthermore, the activity of CARM1 is altered when protein-protein interactions have been documented. it is associated with this complex or with Brg1; whereas free CARM1 has a strong preference for free histone H3 vs. nu- i. Methylation of a transcriptional coactivator. CBP and p300 cleosomal histone H3, the SWI/SNF- or Brg1-associated are essential transcriptional coactivators for a very large CARM1 preferentially methylates nucleosomal histone H3. number of transcription factors, such as cAMP response el- Thus, it appears that the ATP-dependent chromatin remod- ement binding protein (CREB), signal transducer and acti- eling activity helps CARM1 to gain access to otherwise in- vator of transcription 1 (STAT1), activator protein-1 and nu- accessible histone H3 in nucleosomes. The mechanism by clear receptors (181). CBP and p300 are produced from which CARM1 stimulates SWI/SNF activity is unknown, but distinct genes but have a very similar organization of ho- the idea of coordination between these two activities is at- mologous functional domains including a bromodomain, a tractive, given that both are involved in the chromatin re- KIX domain for binding CREB, a protein acetyltransferase modeling process. Another possible link between the SWI/ domain, and three C/H domains that interact with multiple SNF complex and CARM1 was suggested by the recent transcription factors. Arginine methylation of CBP/p300 by discovery that the protein Flightless I can bind to p160 co- CARM1 has been described in two distinct regions. Meth- activators, CARM1, and two components of the SWI/SNF ylation in the KIX domain (amino acids 582–672), observed complex, Brg1 and the actin-like protein BAF53 (178). Flight- in vitro and in vivo, by CARM1 inhibits CREB binding to the less I serves as a coactivator for nuclear receptors in collab- KIX domain (31). Moreover, CARM1 overexpression inhib- oration with p160 coactivators and CARM1. The ability of ited CREB-mediated transcription. It was suggested that the Flightless I to bridge the p160 coactivator complex and the recruitment of CARM1 by nuclear receptors and p160 coac- SWI/SNF complex could possibly contribute to coordination tivators caused this methylation of CBP/p300 and thus pre- of various chromatin remodeling activities, i.e., ATP-depen- vented possibly limiting amounts of CBP/p300 from being dent and protein/histone acetylation and methylation. sequestered by CREB. Methylation of arginines in another conserved region (R714, R742, and R768) by CARM1 was vi. Possible roles for PRMTs in specific gene repression. Are positively implicated in the coactivator function of CBP with PRMTs and arginine-specific protein methylation always as- nuclear receptors (182). Mutations of these arginine residues sociated with active transcription, or can they sometimes to alanine reduced the cooperative coactivator function of contribute to gene repression? PRMT5 was found on a c-myc CBP with GRIP1 and for steroid hormone-induced gene ac- target gene in association with Brg1 and hBrm (which are tivation, but had no effect on CREB-dependent transactiva- alternative ATPase subunits of two related but distinct types tion or retinoic acid response. The authors suggested that this of hSWI/SNF complexes) and with transcriptional corepres- methylation may play a role in CBP coactivator function with sor subunits mSin3A and histone deacetylase 2. This asso- some nuclear receptors and in the synergistic cooperation ciation appeared to be correlated with repression of the target between CBP/p300, p160 coactivators, and CARM1 reported gene (174). PRMT5 was also implicated in repression of the cyclin E promoter (179). It seems likely that some type of previously (162, 163). chromatin remodeling is required for repression as well as ii. Methylation of a DNA-binding transcription factor. Inter- activation of transcription, and that certain types of chro- feron ␣/␤ receptor activation triggers tyrosine and serine matin remodeling activities (e.g., ATP-dependent remodel- phosphorylation of the transcription factor STAT1, leading to ing) may participate in both processes. In addition, p160 STAT1 dimerization and translocation to the nucleus where coactivators, which have primarily been studied in connec- it binds specific enhancer elements and activates transcription with transcriptional activation by nuclear receptors and tion of early interferon response genes. Protein inhibitor of various other transcriptional activators, have been found to activated STAT1 (PIAS1) is a known inhibitor of STAT1 and be involved in hormone-dependent repression of activator exerts its inhibition by binding to the N-terminal region of protein-1 activity by the glucocorticoid receptor (180). Thus, activated STAT1 and thereby preventing STAT1 from bind- it would not be surprising to find that p160-binding second- ing to DNA. STAT1 was recently found to be methylated ary cofactors, such as the PRMTs, sometimes participate in within its N-terminal region by PRMT1 at Arg-31 (183). In- repression as well. Although there is currently no precedent, hibition of STAT1 methylation, by the general AdoMet- one can speculate that the protein substrates for methylation dependent methylation inhibitor 5Ј-methyl-thioadenosine by PRMTs could be different under conditions of transcrip- (MTA) increased PIAS1 binding to STAT1 and reduced tional activation vs. transcriptional repression. STAT1 binding to DNA after interferon ␣ stimulation, re- b. Arginine methylation of nonhistone proteins involved in tran- sulting in a decrease of STAT1 transcriptional activity. Sub- scription and posttranscriptional events. Although histones stitution of Arg-31 with either alanine or glutamic acid also were the first arginine methylation substrates to be associ- resulted in an increase of interferon ␣-mediated transcrip- ated with transcriptional regulation, some earlier studies in tion. Thus, it is possible that Arg-31 is part of the PIAS1 yeast and more recent studies in mammalian cells have be- binding site of STAT1, and that either methylation or mu- gun to identify a variety of protein methylation substrates tation of Arg-31 prevents binding of the inhibitor PIAS1. functioning at various levels of gene regulation: transcription Altogether, these data indicate that STAT1 methylation con- initiation and elongation; and various aspects of RNA me- tributes to the regulation of STAT1 transcriptional activity. 162 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

iii. Regulation of transcription elongation machinery. Tran- hanced Np13p export from the nucleus (4). Some mamma- scriptional elongation factor SPT5 can be methylated in its lian RNA binding proteins are also substrates for arginine RNA pol II binding domain by PRMT1 and PRMT5, resulting methyltransferases. The poly(A)-binding proteins I and II are in inhibition of the elongation-promoting activity of SPT5 methylated by CARM1 (189) and PRMT1 (147), respectively, (184). SPT5 facilitates transcription elongation through its but the functional effect of these methylations is not known. association with both the unphosphorylated and phosphor- The cellular localization of hnRNP A2, which is also methylated forms of RNA pol II. Overexpression of PRMT1 or ylated by PRMT1, is shifted from the nucleus to the cyto- PRMT5 inhibited the ability of Tat to enhance HIV-1 gene plasm upon methyltransferase inhibition, indicating that expression, and drugs that inhibit AdoMet-dependent meth- arginine methylation may promote hnRNP A2 nuclear lo- ylation reversed the inhibition by PRMT1 and PRMT5. In calization (190). The Sam 68 RNA binding protein is meth- addition, mutation of the substrate Arg residues of SPT5 to ylated in vivo by PRMT1; deletion of the methylation sites or Ala or Lys resulted in increased association of SPT5 with its use of methylation inhibitor caused accumulation of Sam 68 target promoters and RNA polymerase and enhancement of in the cytoplasm and prevented Sam 68-mediated export of SPT5 elongation-promoting activity, suggesting that SPT5 HIV RNAs (191). methylation regulates its interaction with the polymerase The mRNA-stabilizing protein HuR was shown to un- and therefore its transcriptional elongation properties. dergo increased methylation within its nucleocytoplasmic Chromatin immunoprecipitation experiments showed that shuttling sequence by CARM1 in response to lipopolysac- PRMT1 and PRMT5 are associated with cytokine-inducible charide (LPS) treatment of a macrophage cell line (148). In- promoters (IL-8 and I␬B␣) under basal conditions but not terestingly, LPS treatment of macrophages leads to HuR- after treatment with the inducer TNF␣, whereas SPT5 is mediated stabilization of TNF␣ mRNA; HuR has been recruited after stimulation with TNF␣. previously implicated in this process, but how LPS regulates ␣ It is worth noting that whereas amino acid substitution at the binding of HuR to the TNF mRNA is not known. Meth- the sites of arginine methylation is a very useful technique, ylation of HuR could enhance its mRNA binding, as was any resulting change in the activity of the protein could shown for methylation of hnRNP A1 by PRMT1 (192, 193). indicate a requisite role for arginine methylation or, alter- On the other hand, because HuR is primarily nuclear and the natively, could indicate that the unmethylated arginine res- methylation occurs within the known shuttling signal, meth- idue is important for function. Thus, caution must be exer- ylation could cause increased cytoplasmic localization of ␣ cised in interpreting such experiments, and corroborating HuR to facilitate its binding to the cytoplasmic TNF mRNA. evidence from other types of experiments is needed for a Changes in intracellular localization caused by methylation clear interpretation of the mutant phenotype. could modify protein-protein interactions between RNA binding proteins like HuR and proteins such as pp32 and iv. Regulation of RNA processing and export. Involvement of APRIL that mediate nuclear export (194). arginine methylation in posttranscriptional events is illus- trated by studies with the small nuclear ribonucleoprotein 2. Arginine methylation in signal transduction. Like phosphor- particles (snRNPs) SmD1 and SmD3, which constitute the ylation, protein methylation is used as a signaling mecha- common core of the spliceosomal snRNPs. SmD1 and SmD3 nism. In various signaling pathways, arginine-specific pro- require symmetric dimethylation of arginine to bind effi- tein methylation can alter the shape or stability of a protein, ciently to the survival of motor neuron (SMN) protein, in- and arginine methylation can promote or inhibit specific volved in spinal muscular atrophy (185). SMN functions as intermolecular interactions. For example, when nuclear re- a chaperone of large macromolecular complexes by assem- ceptors activate transcription, as discussed in detail in Section bling the Sm proteins on snRNA to form an snRNP core III.B.1, PRMT-mediated methylation of histones and other particle. The core particle is responsible for snRNA nuclear protein components of the transcription machinery is part of transport and mRNA cap hypermethylation. Thus, by reg- a signal transduction pathway that transmits the activating ulating the binding of Sm proteins to SMN, a yet-to be iden- signal from the hormone-activated, DNA-bound nuclear re- tified type II PRMT regulates RNP assembly. As the only ceptor to the chromatin and transcription machinery. PRMTs currently known type II enzyme, PRMT5 must be considered are also implicated in a wide range of other signaling path- a candidate. Moreover, PRMT5 has been found in a complex ways that regulate transcription. PRMT1 can bind to the with Sm proteins (186). cytoplasmic domain of the interferon ␣/␤ receptor, and cells From synthesis to translation, mRNA is complexed with deficient in the methyltransferase are more resistant to heterogeneous nuclear ribonucleoproteins (hnRNPs), which growth inhibition by interferon (140). The subsequent ob- among other functions mediate mRNA export from the nu- servation that STAT1 is methylated by PRMT1 in response to cleus into the cytoplasm. Many hnRNPs have long been interferon (183) suggests that PRMT1 might be recruited by known to be methylated on arginine residues, particularly the activated interferon receptor to methylate STAT1. The within the Arg-Gly-Gly repeats that are often associated with methylation of STAT1 appears to complement the phosphor- RNA-binding motifs (146, 187). In the yeast S. cerevisiae, ylation of STAT1 by Jak kinases, which also occurs as a result Hmt1/Rmt1 methylates the hnRNPs Np13p, Hrp1p (4), and of interferon receptor activation; phosphorylation potenti- Nab2p (188), which shuttle between nucleus and cytoplasm ates the nuclear translocation of STAT1 and its binding to its in their role in facilitating mRNA export from the nucleus. cognate enhancer element, whereas methylation prevents Nuclear export of these hnRNPs was defective in cells lack- binding of the inhibitory protein PIAS1. Another PRMT im- ing the methyltransferase, and overexpression of Hmt1 en- plicated in the same signal transduction pathway is PRMT5, Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 163 which was first identified as a Jak-kinase binding protein Hormones regulate many cellular processes by triggering (143). Jak kinases are involved in cytokine- as well as inter- signal transduction pathways that result in a variety of post- feron-induced signaling, and in fact both PRMT1 and PRMT5 translational modifications to proteins. Although protein have been found to associate with cytokine-inducible phosphorylation is the best characterized modification, it is promoters (184). In addition to its role in interferon- and clear from the accumulated work discussed above that meth- cytokine-induced Jak-STAT signaling, PRMT1 has also been ylation on arginine and lysine residues also functions as a implicated in mitogen-activated signaling. The first cloning component in many signaling pathways. Thus, the methyl- of PRMT1 resulted from its ability to bind TIS21 and BTG1, ation event must be regulated by upstream activity in the which are immediate-early proteins induced by mitogen signaling pathway, and it must have specific consequences treatment, such as nerve growth factor (NGF) stimulation of that propagate the signal to downstream components. At PC12 cells (139). NGF also causes increased PRMT1 activity present, we have identified a substantial number of meth- in PC12 cells and thus induces the methylation of several ylated proteins and enzymes that make such modifications, specific (unidentified) proteins during neuronal differentia- but we still have much to learn about how the methylation tion of PC12 cells (195, 196). events are regulated and the specific molecular and physiological consequences of this modification. C. Regulation of arginine methyltransferase activity How are the specificity and regulation of these methylation events achieved? The answer is probably 2-fold. The If arginine methylation of proteins serves as a signaling substrate specificity of the enzymes themselves will obvi- mechanism, then it must be regulated in some manner. The ously define the potential substrates. In addition, evidence to enzymatic activity of a PRMT could be modulated by post- date indicates that the availability of the substrate to the translational modifications or protein-protein interactions; enzyme is also regulated. For example, although histone or the access of the enzyme to the substrate could be regu- methyltransferases are constantly present in cells, the specific lated. There are hints that all three of these mechanisms could chromatin regions and nucleosomes that are methylated are be used in different situations. PRMT6 (145), PRMT1, and determined by specific recruitment of the HMTs and PRMTs CARM1 (S. S. Koh, C. Teyssier, H. Li, and M. R. Stallcup, by DNA binding transcription factors, small RNA species unpublished observations) display automethylation activity, associated with certain regions of chromatin, RNA polymer- although the effects are still unknown. Binding of the mito- ase, or certain types of histone modifications (Figs. 2 and 4). gen response immediate-early proteins, TIS21 and its ho- Does a particular histone methylation event (i.e., at a spe- molog BTG1, to PRMT1 modulates PRMT1 activity (139). cific Arg or Lys residue of a specific histone) always have the Because formation of homodimers or larger homooligomers same consequences? It is too early to know for sure. The has also been linked to enzyme activity for Hmt1 (149), correlations of specific methylation events with active or PRMT1 (133), and PRMT5 (144), regulation of multimer for- repressed transcription are pretty strong at this time, but mation could conceivably serve as a means of regulating there are hints that differences may occur, as indicated by our PRMT enzymatic activity. Controlled access to substrate ap- discussion of evidence for both activation and repression pears to be important in the case of histone arginine meth- associated with several specific lysine methylation events. ylation by CARM1 and PRMT1 during nuclear receptor- Another example involves HP1, which binds histone H3 mediated transcriptional activation, because chromatin methylated at Lys-9. Although HP1 is primarily associated immunoprecipitation assays show hormone-dependent as- with heterochromatin, genetic experiments in Drosophila sug- sociation of the PRMTs and arginine methylation of histones gest that it may be involved in activation of a few genes (112). specifically at the hormonally regulated promoters (164, 165). We know that different promoters occur in different chro- There are two cases where evidence of enhanced PRMT matin conformation and DNA sequence contexts and recruit activity is observed. Extracts of NGF-treated PC12 cells ex- different combinations of transcriptional regulatory factors hibit elevated levels of PRMT1 activity, compared with ex- and complexes. In addition, multiple signaling pathways tracts from untreated cells (195); the mechanism of enhance- presumably converge to regulate simultaneously the tran- ment is still unclear. On the other hand, LPS treatment of scription of a particular gene, and these signals must be macrophage cells leads to enhanced methylation of HuR integrated into a single action, i.e., determining the efficiency protein by CARM1 (148), but no increase in the level of of producing mRNA from that gene. Thus, it seems likely CARM1 or its activity was detected in cell extracts (S. S. Koh, that the various interacting regulatory components can in- C. Teyssier, H. Li, and M. R. Stallcup, unpublished obser- fluence each other’s activities, so that a specific type of hi- vations), suggesting that substrate access might be regulated stone modification may have different effects in different in some way. Clearly, additional work is needed to elucidate regulatory contexts. such mechanisms. B. Are arginine and lysine methylation reversible or stable marks? IV. Conclusions and Unanswered Questions A. Protein methylation and endocrinology Whether arginine and lysine methylation can be removed remains an open question. In contrast to phosphorylation or Although many questions remain to be answered, it is clear acetylation, no enzyme capable of removing a methyl group that methylation of lysine and arginine residues of proteins has been found so far, leading to the hypothesis that arginine plays widespread and important roles in endocrinology. and lysine methylation might be irreversible and that these 164 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription modifications could be present during the whole life of a inactive X, suggesting that this modification is not stably protein substrate. However, Annunziato et al. (197), using inherited and is somehow removed as the X inactivation pulse-labeling studies in HeLa cells, have shown that histone process continues (114, 115). H3 methylation still occurred in cell cycle-arrested cells when Examples of dynamic methylation of H3 Lys-4 also exist. histone synthesis was lowered. They concluded that the ob- In S. cerevisiae, a time-course study showed that upon served H3 methylation was due to methyl group turnover GAL10 gene induction, there was a rapid recruitment of rather than new histone synthesis. A similar case was made RNA Pol II and Set1 that coincided with an increase in di- for association of dynamically methylated histones H3 and and trimethylation of H3 Lys-4. When the gene was turned H4 with active (i.e., acetylated) chromatin in chicken eryth- off by switching to a repressive medium containing glu- rocytes (198). Although these two studies did not distinguish cose, RNA Pol II and Set1 were released from the 5Ј coding between lysine and arginine methylation and were done on region of the gene immediately, but methylation of H3 bulk histones rather than at specific promoters, these data Lys-4 persisted and then returned to baseline levels after suggest that histone methylation is a dynamic process that 6 h (45). This led to the proposal of “recent transcriptional occurs even in the absence of new histone synthesis. Earlier memory,” meaning methylation of histones could mark studies with Drosophila cells found that heat shock led to genes that were recently active. Another example of acute decreased lysine methylation and increased arginine meth- reduction in the level of H3 Lys-4 methylation involved a ylation of bulk histone H3 (199). With the availability of steroid hormone inducible promoter in mammalian cul- highly specific antibodies for histones methylated at specific lysines or arginines, recent reports describe changes in the tured cells. Activation of PSA gene transcription by an- level of arginine and lysine methylation of histones during drogen hormone led to reduced H3 Lys-4 methylation in gene activation, at different stages of cell cycle, and in the the promoter region, but an increase in this modification initial phase of X inactivation. In human dendritic cells, in the coding region (203). In addition, a recent chromatin which are postmitotic and terminally differentiated, treat- immunoprecipitation analysis indicated that estradiol ment with LPS led to rapid induction of ELC, MDC and treatment of MCF-7 cells leads to cyclical appearance and IL-12p40 genes (200). The promoters of these genes contain disappearance of histone methylation at the pS2 promoter. dimethylated H3 Lys-9 under unstimulated conditions, but Appearance and disappearance of H3 Arg-17 methylation upon LPS treatment the dimethylation level of H3 Lys-9 had a 40-min cycle, whereas H4 Arg-3 methylation had an decreased concomitant with recruitment of RNA Pol II. 80-min cycle (168). When RNA Pol II was released from the promoter after 24 to Although all of the above examples involve changes in 72 h, dimethylation of H3 Lys-9 was restored to the unstimu- histones from a methylated to unmethylated state, the mech- lated level. In experiments utilizing Xenopus oocytes to as- anism of this change is unclear. Histone methylation could semble chromatin from microinjected DNA templates, it was in principle be reversible by demethylation, histone prote- shown that promoters regulated by thyroid hormone and its olysis, or histone replacement, independent of new global nuclear receptor contained significant dimethylated H3 histone synthesis during S-phase. In Tetrahymena, the first six Lys-9 when occupied by unliganded thyroid hormone re- amino acids of histone H3 are cleaved to form a Hf (fast ceptor (201). Overnight thyroid hormone treatment caused a migrating histone) in transcriptionally inactive micronuclei decrease in the level of dimethylated H3 Lys-9 and an in- (204). Although such an occurrence has not been actively crease in the level of methylated H3 Lys-4. Thus, these two investigated in higher eukaryotes, it is interesting to note that examples show that methylation of histone H3 at Lys-9 proteolysis of the first six amino acids would remove meth- can be dynamically regulated, although the mechanism ylated H3 Lys-4 and provide a new epitope for substrate for reverting methylated to unmethylated histone is still recognition. unknown. Possible mechanisms for removal and replacement of Methylation levels of H4 Lys-20 and Arg-3 are cell cycle some histones during transcription have been previously regulated. Western blot analysis shows that the level of described (12), and the preferential association of the hi- methylated Lys-20 decreases during the late S phase and stone H3.3 variant with transcriptionally active chromatin increases back to normal level at mitosis. Methylated (205) also suggests that histone replacement could occur in Arg-3, on the other hand, decreases during early S phase and comes back up to normal levels during late S phase connection with transcription. Histone H3.3 is synthesized (202). Fluctuation of H3 Lys-79 methylation is similar to throughout the cell cycle and is able to replace histone H3 that of H4 Lys-20 methylation (75). Although demethyl- independent of DNA replication. Such a replacement ation could be responsible, the decrease in the methylation mechanism would allow resetting of modifications present on level observed during S phase could simply be due to the the N-terminal tails of histone H3. A relatively stable level of deposition of new histones during DNA replication, lead- histone methylation could also constitute a type of molecular ing to dilution of methylated histones. On the other hand, memory of previous activity at a specific gene. However, the disappearance of EED-EZH2-dependent trimethyla- whether and how such modifications could be faithfully trans- tion of H3 Lys-27 is less easily explained by replication- mitted during or after DNA replication is unclear and will dependent deposition of new histones. During the initial require further investigation. Possible mechanisms for demeth- stage of X chromosome inactivation, trimethylation of H3 ylation or otherwise reversing histone methylation and the con- Lys-27 increases steadily and reaches a peak at d 6. By d cept of histone methylation as a stable mark have been dis- 13, trimethylated H3 Lys-27 completely disappears from cussed in a recent review (206). Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 165

C. Future directions 8. Luger K, Ma¨der AW, Richmond RK, Sargent DF, Richmond TJ 1997 Crystal structure of the nucleosome core particle at 2.8 Å resolution. Although protein arginine methylation is now clearly in- Nature 389:251–260 volved at many levels of gene regulation and signal trans- 9. Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P, Denu JM, duction, the mechanisms by which protein methylation con- Allis CD 2000 Synergistic coupling of histone H3 phosphorylation tributes to these physiological processes (i.e., the functional and acetylation in response to epidermal growth factor stimulation. Mol Cell 5:905–915 ramifications of protein methylation) are still mostly un- 10. Lo W-S, Trievel RC, Rojas JR, Duggan L, Hsu J-Y, Allis CD, known. As has been the case with protein phosphorylation, Marmorstein R, Berger SL 2000 Phosphorylation of serine 10 in PRMTs will undoubtedly continue to be discovered as par- histone H3 is functionally linked in vitro and in vivo to Gcn5- ticipants in a variety of additional cellular processes and mediated acetylation at lysine 14. Mol Cell 5:917–926 11. Stallcup MR 2001 Role of protein methylation in chromatin re- signaling pathways in the next few years, and many new modeling and transcriptional regulation. Oncogene 20:3014–3020 substrates for these enzymes probably remain to be identi- 12. Zhang Y, Reinberg D 2001 Transcription regulation by histone fied. S. cerevisiae and mammalian systems aside, the identities methylation: interplay between different covalent modifications of and roles of PRMTs in most other popular experimental the core histone tails. Genes Dev 15:2343–2360 organisms are yet to be determined, and it would not be 13. Fischle W, Wang Y, Allis CD 2003 Binary switches and modification cassettes in histone biology and beyond. Nature 425:475–479 completely surprising if a few additional members of the 14. Berger SL 2002 Histone modifications in transcriptional regulation. mammalian PRMT family are found. Finally, the question of Curr Opin Genet Dev 12:142–148 reversibility of protein arginine and lysine methylation re- 15. Zhang L, Eugeni EE, Parthun MR, Freitas MA 2003 Identification mains to be solved. of novel histone post-translational modifications by peptide mass More challenging work also lies ahead in the field of lysine fingerprinting. Chromosoma 112:77–86 16. Zhang K, Tang H, Huang L, Blankenship JW, Jones PR, Xiang F, methylation. Work with HP1 and Pc has established the Yau PM, Burlingame AL 2002 Identification of acetylation and principle that there are proteins which bind preferentially to methylation sites of histone H3 from chicken erythrocytes by high- histones methylated at specific residues and interpret the accuracy matrix-assisted laser desorption ionization-time-of-flight, methylation signal into a specific biological response. Iden- matrix-assisted laser desorption ionization-postsource decay, and tification of additional proteins that preferentially bind other nanoelectrospray ionization tandem mass spectrometry. Anal Bio- chem 306:259–269 methylated lysine and arginine residues of histones will help 17. Zhang X, Tamaru H, Khan SI, Horton JR, Keefe LJ, Selker EU, decipher the “histone code.” Confusion regarding mecha- Cheng X 2002 Structure of the Neurospora SET domain protein nisms of targeting at the global and gene-specific level will DIM-5, a histone H3 lysine methyltransferase. Cell 111:117–127 hopefully be reconciled with further study. Finally, it seems 18. Wilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackburn inevitable that many more nonhistone substrates of lysine GM, Gamblin SJ, Xiao B 2002 Crystal structure and functional analysis of the histone methyltransferase SET7/9. Cell 111:105–115 methylation will be discovered very soon. 19. Trievel RC, Beach BM, Dirk LM, Houtz RL, Hurley JH 2002 Structure and catalytic mechanism of a SET domain protein methyltransferase. Cell 111:91–103 Acknowledgments 20. Zhang X, Yang Z, Khan SI, Horton JR, Tamaru H, Selker EU, Cheng X 2003 Structural basis for the product specificity of histone Address all correspondence and requests for reprints to: Michael R. lysine methyltransferases. Mol Cell 12:177–185 Stallcup, Department of Pathology, HMR 301, University of Southern 21. Xiao B, Jing C, Wilson JR, Walker PA, Vasisht N, Kelly G, Howell California, 2011 Zonal Avenue, Los Angeles, California 90089-9092. S, Taylor IA, Blackburn GM, Gamblin SJ 2003 Structure and E-mail: [email protected] catalytic mechanism of the human histone methyltransferase This work was supported by awards from the National Institutes of SET7/9. Nature 421:652–656 Health (NIH) (DK55274 to M.R.S. and GM068088 to B.D.S.). B.D.S. is a 22. Min J, Zhang X, Cheng X, Grewal SI, Xu RM 2002 Structure of the Pew Scholar in the Biomedical Sciences. D.Y.L. was supported by a SET domain histone lysine methyltransferase Clr4. Nat Struct Biol predoctoral fellowship from NIH training grant DE07211. 9:828–832 23. Kwon T, Chang JH, Kwak E, Lee CW, Joachimiak A, Kim YC, Lee J, Cho Y 2003 Mechanism of histone lysine methyl transfer revealed References by the structure of SET7/9-AdoMet. EMBO J 22:292–303 24. van Leeuwen F, Gafken PR, Gottschling DE 2002 Dot1p modu- 1. Strahl BD, Allis CD 2000 The language of covalent histone mod- lates silencing in yeast by methylation of the nucleosome core. Cell ifications. Nature 403:41–45 109:745–756 2. Jenuwein T, Allis CD 2001 Translating the histone code. Science 25. Strahl BD, Grant PA, Briggs SD, Sun ZW, Bone JR, Caldwell JA, 293:1074–1080 Mollah S, Cook RG, Shabanowitz J, Hunt DF, Allis CD 2002 Set2 3. Aletta JM, Cimato TR, Ettinger MJ 1998 Protein methylation: a is a nucleosomal histone H3-selective methyltransferase that me- signal event in post-translational modification. Trends Biochem Sci diates transcriptional repression. Mol Cell Biol 22:1298–1306 23:89–91 26. Nishioka K, Rice JC, Sarma K, Erdjument-Bromage H, Werner J, 4. Shen EC, Henry MF, Weiss VH, Valentini SR, Silver PA, Lee MS Wang Y, Chuikov S, Valenzuela P, Tempst P, Steward R, Lis JT, 1998 Arginine methylation facilitates the nuclear export of hnRNP Allis CD, Reinberg D 2002 PR-Set7 is a nucleosome-specific meth- proteins. Genes Dev 12:679–691 yltransferase that modifies lysine 20 of histone H4 and is associated 5. van Holde KE 1989 Chromatin. New York: Springer-Verlag; with silent chromatin. Mol Cell 9:1201–1213 111–148 27. Fang J, Feng Q, Ketel CS, Wang H, Cao R, Xia L, Erdjument- 6. Chen D, Ma H, Hong H, Koh SS, Huang S-M, Schurter BT, Aswad Bromage H, Tempst P, Simon JA, Zhang Y 2002 Purification and DW, Stallcup MR 1999 Regulation of transcription by a protein functional characterization of SET8, a nucleosomal histone H4- methyltransferase. Science 284:2174–2177 lysine 20-specific methyltransferase. Curr Biol 12:1086–1099 7. Strahl BD, Ohba R, Cook RG, Allis CD 1999 Methylation of 28. Grant PA, Duggan L, Cote J, Roberts SM, Brownell JE, Candau histone H3 at lysine 4 is highly conserved and correlates with R, Ohba R, Owen-Hughes T, Allis CD, Winston F, Berger SL, transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci Workman JL 1997 Yeast Gcn5 functions in two multisubunit com- USA 96:14967–14972 plexes to acetylate nucleosomal histones: characterization of an 166 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

Ada complex and the SAGA (Spt/Ada) complex. Genes Dev 11: 49. Sun ZW, Allis CD 2002 Ubiquitination of histone H2B regulates H3 1640–1650 methylation and gene silencing in yeast. Nature 418:104–108 29. Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun Z-W, Schmid 50. Briggs SD, Xiao T, Sun ZW, Caldwell JA, Shabanowitz J, Hunt M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T 2000 DF, Allis CD, Strahl BD 2002 Gene silencing: trans-histone reg- Regulation of chromatin structure by site-specific histone H3 meth- ulatory pathway in chromatin. Nature 418:498 yltransferases. Nature 406:593–599 51. Ng HH, Xu RM, Zhang Y, Struhl K 2002 Ubiquitination of histone 30. Gu W, Roeder RG 1997 Activation of p53 sequence-specific DNA H2B by Rad6 is required for efficient Dot1-mediated methylation binding by acetylation of the p53 C-terminal domain. Cell 90: of histone H3 lysine 79. J Biol Chem 277:34655–34657 595–606 52. Wood A, Krogan NJ, Dover J, Schneider J, Heidt J, Boateng MA, 31. Xu W, Chen H, Du K, Asahara H, Tini M, Emerson BM, Mont- Dean K, Golshani A, Zhang Y, Greenblatt JF, Johnston M, Shi- miny M, Evans RM 2001 A transcriptional switch mediated by latifard A 2003 Bre1, an E3 ubiquitin ligase required for recruitment cofactor methylation. Science 294:2507–2511 and substrate selection of Rad6 at a promoter. Mol Cell 11:267–274 32. Chen H, Lin RJ, Xie W, Wilpitz D, Evans RM 1999 Regulation of 53. Hwang WW, Venkatasubrahmanyam S, Ianculescu AG, Tong A, hormone-induced histone hyperacetylation and gene activation via Boone C, Madhani HD 2003 A conserved RING finger protein acetylation of an acetylase. Cell 98:675–686 required for histone H2B monoubiquitination and cell size control. 33. Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM, Mol Cell 11:261–266 Mullen T-M, Glass CK, Rosenfeld MG 1998 Transcription factor- 54. Ng HH, Dole S, Struhl K 2003 The Rtf1 component of the Paf1 specific requirements for coactivators and their acetyltransferase transcriptional elongation complex is required for ubiquitination of functions. Science 279:703–707 histone H2B. J Biol Chem 278:33625–33628 34. Richards EJ, Elgin SC 2002 Epigenetic codes for heterochromatin 55. Wood A, Schneider J, Dover J, Johnston M, Shilatifard A 2003 The formation and silencing: rounding up the usual suspects. Cell 108: Paf1 complex is essential for histone monoubiquitination by the 489–500 Rad6-Bre1 complex, which signals for histone methylation by 35. Weiler KS, Wakimoto BT 1995 Heterochromatin and gene expres- COMPASS and Dot1p. J Biol Chem 278:34739–34742 sion in Drosophila. Annu Rev Genet 29:577–605 56. Xiao T, Kao CF, Krogan NJ, Sun ZW, Greenblatt JF, Osley MA, 36. Briggs SD, Bryk M, Strahl BD, Cheung WL, Davie JK, Dent SY, Strahl BD 2005 Histone H2B ubiquitylation is associated with Winston F, Allis CD 2001 Histone H3 lysine 4 methylation is elongating RNA polymerase II. Mol Cell Biol 25:637–651 mediated by Set1 and required for cell growth and rDNA silencing 57. Ezhkova E, Tansey WP 2004 Proteasomal ATPases link ubiquity- in Saccharomyces cerevisiae. Genes Dev 15:3286–3295 lation of histone H2B to methylation of histone H3. Mol Cell 13: 37. Noma K, Allis CD, Grewal SI 2001 Transitions in distinct histone 435–442 H3 methylation patterns at the heterochromatin domain bound- 58. Roguev A, Schaft D, Shevchenko A, Pijnappel WW, Wilm M, aries. Science 293:1150–1155 Aasland R, Stewart AF 2001 The Saccharomyces cerevisiae Set1 com- 38. Litt MD, Simpson M, Gaszner M, Allis CD, Felsenfeld G 2001 plex includes an Ash2 homologue and methylates histone 3 lysine Correlation between histone lysine methylation and developmen- 4. EMBO J 20:7137–7148 tal changes at the chicken ␤-globin locus. Science 293:2453–2455 59. Nagy PL, Griesenbeck J, Kornberg RD, Cleary ML 2002 A tritho- 39. Morshead KB, Ciccone DN, Taverna SD, Allis CD, Oettinger MA rax-group complex purified from Saccharomyces cerevisiae is re- 2003 Antigen receptor loci poised for V(D)J rearrangement are quired for methylation of histone H3. Proc Natl Acad Sci USA broadly associated with BRG1 and flanked by peaks of histone H3 99:90–94 dimethylated at lysine 4. Proc Natl Acad Sci USA 100:11577–11582 60. Sedkov Y, Cho E, Petruk S, Cherbas L, Smith ST, Jones RS, 40. Boa S, Coert C, Patterton HG 2003 Saccharomyces cerevisiae Set1p is Cherbas P, Canaani E, Jaynes JB, Mazo A 2003 Methylation at a methyltransferase specific for lysine 4 of histone H3 and is re- lysine 4 of histone H3 in ecdysone-dependent development of quired for efficient gene expression. Yeast 20:827–835 Drosophila. Nature 426:78–83 41. Bernstein BE, Humphrey EL, Erlich RL, Schneider R, Bouman P, 61. Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD, Liu JS, Kouzarides T, Schreiber SL 2002 Methylation of histone H3 Hess JL 2002 MLL targets SET domain methyltransferase activity Lys 4 in coding regions of active genes. Proc Natl Acad Sci USA to Hox gene promoters. Mol Cell 10:1107–1117 99:8695–8700 62. Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, 42. Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J, Bernstein Wassell R, Dubois G, Mazo A, Croce CM, Canaani E 2002 ALL-1 BE, Emre NC, Schreiber SL, Mellor J, Kouzarides T 2002 Active is a histone methyltransferase that assembles a supercomplex of genes are tri-methylated at K4 of histone H3. Nature 419:407–411 proteins involved in transcriptional regulation. Mol Cell 10:1119– 43. Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane- 1128 Robinson C, Kouzarides T 2004 Histone H3 lysine 4 methylation 63. Wysocka J, Myers MP, Laherty CD, Eisenman RN, Herr W 2003 patterns in higher eukaryotic genes. Nat Cell Biol 6:73–77 Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone 44. Liang G, Lin JC, Wei V, Yoo C, Cheng JC, Nguyen CT, Weisen- H3–K4 methyltransferase are tethered together selectively by the berger DJ, Egger G, Takai D, Gonzales FA, Jones PA 2004 Distinct cell-proliferation factor HCF-1. Genes Dev 17:896–911 localization of histone H3 acetylation and H3–K4 methylation to the 64. Wang H, Cao R, Xia L, Erdjument-Bromage H, Borchers C, transcription start sites in the human genome. Proc Natl Acad Sci Tempst P, Zhang Y 2001 Purification and functional characteriza- USA 101:7357–7362 tion of a histone H3-lysine 4-specific methyltransferase. Mol Cell 45. Ng HH, Robert F, Young RA, Struhl K 2003 Targeted recruitment 8:1207–1217 of Set1 histone methylase by elongating Pol II provides a localized 65. Nishioka K, Chuikov S, Sarma K, Erdjument-Bromage H, Allis mark and memory of recent transcriptional activity. Mol Cell 11: CD, Tempst P, Reinberg D 2002 Set9, a novel histone H3 methyl- 709–719 transferase that facilitates transcription by precluding histone tail 46. Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, Boateng MA, modifications required for heterochromatin formation. Genes Dev Dean K, Ryan OW, Golshani A, Johnston M, Greenblatt JF, 16:479–489 Shilatifard A 2003 The Paf1 complex is required for histone H3 66. Nislow C, Ray E, Pillus L 1997 SET1, a yeast member of the methylation by COMPASS and Dot1p: linking transcriptional elon- trithorax family, functions in transcriptional silencing and diverse gation to histone methylation. Mol Cell 11:721–729 cellular processes. Mol Biol Cell 8:2421–2436 47. Komarnitsky P, Cho EJ, Buratowski S 2000 Different phosphor- 67. Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, ylated forms of RNA polymerase II and associated mRNA pro- Popkin D, Pillus L, Jenuwein T 1997 Mammalian homologues of cessing factors during transcription. Genes Dev 14:2452–2460 the polycomb-group gene enhancer of zeste mediate gene silencing 48. Cho EJ, Kobor MS, Kim M, Greenblatt J, Buratowski S 2001 in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of J 16:3219–3232 the RNA polymerase II C-terminal domain. Genes Dev 15:3319– 68. Bryk M, Briggs SD, Strahl BD, Curcio MJ, Allis CD, Winston F 3329 2002 Evidence that Set1, a factor required for methylation of histone Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 167

H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent presses basal expression of GAL4 in Saccharomyces cerevisiae. Mol mechanism. Curr Biol 12:165–170 Cell Biol 23:5972–5978 69. Huang H, Kahana A, Gottschling DE, Prakash L, Liebman SW 89. Eissenberg JC, James TC, Foster-Hartnett DM, Hartnett T, Ngan 1997 The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for V, Elgin SC 1990 Mutation in a heterochromatin-specific chromo- silencing in Saccharomyces cerevisiae. Mol Cell Biol 17:6693–6699 somal protein is associated with suppression of position-effect 70. van Leeuwen F, Gottschling DE 2002 Genome-wide histone mod- variegation in Drosophila melanogaster. Proc Natl Acad Sci USA ifications: gaining specificity by preventing promiscuity. Curr Opin 87:9923–9927 Cell Biol 14:756–762 90. James TC, Elgin SC 1986 Identification of a nonhistone chromo- 71. Suka N, Suka Y, Carmen AA, Wu J, Grunstein M 2001 Highly somal protein associated with heterochromatin in Drosophila mela- specific antibodies determine histone acetylation site usage in yeast nogaster and its gene. Mol Cell Biol 6:3862–3872 heterochromatin and euchromatin. Mol Cell 8:473–479 91. Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, 72. Noma K, Grewal SI 2002 Histone H3 lysine 4 methylation is me- Allshire RC, Kouzarides T 2001 Selective recognition of methyl- diated by Set1 and promotes maintenance of active chromatin ated lysine 9 on histone H3 by the HP1 chromo domain. Nature states in fission yeast. Proc Natl Acad Sci USA 99(Suppl 4):16438– 410:120–124 16445 92. Lachner M, O’Carroll D, Rea S, Mechtler K, Jenuwein T 2001 73. Ng HH, Feng Q, Wang H, Erdjument-Bromage H, Tempst P, Methylation of histone H3 lysine 9 creates a binding site for HP1 Zhang Y, Struhl K 2002 Lysine methylation within the globular proteins. Nature 410:116–120 domain of histone H3 by Dot1 is important for telomeric silencing 93. Rice JC, Briggs SD, Ueberheide B, Barber CM, Shabanowitz J, and Sir protein association. Genes Dev 16:1518–1527 Hunt DF, Shinkai Y, Allis CD 2003 Histone methyltransferases 74. Lacoste N, Utley RT, Hunter JM, Poirier GG, Cote J 2002 Disruptor direct different degrees of methylation to define distinct chromatin of telomeric silencing-1 is a chromatin-specific histone H3 methyl- domains. Mol Cell 12:1591–1598 transferase. J Biol Chem 277:30421–30424 94. Peters AH, Kubicek S, Mechtler K, O’Sullivan RJ, Derijck AA, 75. Feng Q, Wang H, Ng HH, Erdjument-Bromage H, Tempst P, Perez-Burgos L, Kohlmaier A, Opravil S, Tachibana M, Shinkai Struhl K, Zhang Y 2002 Methylation of H3-lysine 79 is mediated Y, Martens JH, Jenuwein T 2003 Partitioning and plasticity of by a new family of HMTases without a SET domain. Curr Biol repressive histone methylation states in mammalian chromatin. 12:1052–1058 Mol Cell 12:1577–1589 76. Ng HH, Ciccone DN, Morshead KB, Oettinger MA, Struhl K 2003 95. Maison C, Bailly D, Peters AH, Quivy JP, Roche D, Taddei A, Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast Lachner M, Jenuwein T, Almouzni G 2002 Higher-order structure and mammalian cells: a potential mechanism for position-effect in pericentric heterochromatin involves a distinct pattern of histone variegation. Proc Natl Acad Sci USA 100:1820–1825 modification and an RNA component. Nat Genet 30:329–334 77. Im H, Park C, Feng Q, Johnson KD, Kiekhaefer CM, Choi K, 96. Tachibana M, Sugimoto K, Fukushima T, Shinkai Y 2001 Set Zhang Y, Bresnick EH 2003 Dynamic regulation of histone H3 domain-containing protein, G9a, is a novel lysine-preferring mam- methylated at lysine 79 within a tissue-specific chromatin domain. malian histone methyltransferase with hyperactivity and specific J Biol Chem 278:18346–18352 selectivity to lysines 9 and 27 of histone H3. J Biol Chem 276: 78. San Segundo PA, Roeder GS 2000 Role for the silencing protein 25309–25317 Dot1 in meiotic checkpoint control. Mol Biol Cell 11:3601–3615 97. Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, 79. Zegerman P, Canas B, Pappin D, Kouzarides T 2002 Histone H3 Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y 2002 G9a lysine 4 methylation disrupts binding of nucleosome remodeling and histone methyltransferase plays a dominant role in euchromatic deacetylase (NuRD) repressor complex. J Biol Chem 277:11621–11624 histone H3 lysine 9 methylation and is essential for early embry- 80. Henry KW, Wyce A, Lo WS, Duggan LJ, Emre NC, Kao CF, Pillus ogenesis. Genes Dev 16:1779–1791 L, Shilatifard A, Osley MA, Berger SL 2003 Transcriptional acti- 98. Nakayama J, Rice JC, Strahl BD, Allis CD, Grewal SI 2001 Role vation via sequential histone H2B ubiquitylation and deubiquity- of histone H3 lysine 9 methylation in epigenetic control of hetero- lation, mediated by SAGA-associated Ubp8. Genes Dev 17:2648– chromatin assembly. Science 292:110–113 2663 99. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen 81. Kao CF, Hillyer C, Tsukuda T, Henry K, Berger S, Osley MA 2004 RA 2002 Regulation of heterochromatic silencing and histone H3 Rad6 plays a role in transcriptional activation through ubiquity- lysine-9 methylation by RNAi. Science 297:1833–1837 lation of histone H2B. Genes Dev 18:184–195 100. Allshire R 2002 Molecular biology. RNAi and heterochromatin—a 82. Daniel JA, Torok MS, Sun ZW, Schieltz D, Allis CD, Yates III JR, hushed-up affair. Science 297:1818–1819 Grant PA 2004 Deubiquitination of histone H2B by a yeast acetyl- 101. Reinhart BJ, Bartel DP 2002 Small RNAs correspond to centromere transferase complex regulates transcription. J Biol Chem 279:1867– heterochromatic repeats. Science 297:1831 1871 102. Akhtar A, Zink D, Becker PB 2000 Chromodomains are protein- 83. Xiao T, Hall H, Kizer KO, Shibata Y, Hall MC, Borchers CH, RNA interaction modules. Nature 407:405–409 Strahl BD 2003 Phosphorylation of RNA polymerase II CTD reg- 103. Jenuwein T 2002 Molecular biology. An RNA-guided pathway for ulates H3 methylation in yeast. Genes Dev 17:654–663 the epigenome. Science 297:2215–2218 84. Li J, Moazed D, Gygi SP 2002 Association of the histone methyl- 104. Nielsen SJ, Schneider R, Bauer UM, Bannister AJ, Morrison A, transferase Set2 with RNA polymerase II plays a role in transcrip- O’Carroll D, Firestein R, Cleary M, Jenuwein T, Herrera RE, tion elongation. J Biol Chem 277:49383–49388 Kouzarides T 2001 Rb targets histone H3 methylation and HP1 to 85. Schaft D, Roguev A, Kotovic KM, Shevchenko A, Sarov M, promoters. Nature 412:561–565 Shevchenko A, Neugebauer KM, Stewart AF 2003 The histone 3 105. Vandel L, Nicolas E, Vaute O, Ferreira R, Ait-Si-Ali S, Trouche lysine 36 methyltransferase, SET2, is involved in transcriptional D 2001 Transcriptional repression by the retinoblastoma protein elongation. Nucleic Acids Res 31:2475–2482 through the recruitment of a histone methyltransferase. Mol Cell 86. Krogan NJ, Kim M, Tong A, Golshani A, Cagney G, Canadien V, Biol 21:6484–6494 Richards DP, Beattie BK, Emili A, Boone C, Shilatifard A, 106. Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher III Buratowski S, Greenblatt J 2003 Methylation of histone H3 by Set2 FJ 2002 SETDB1: a novel KAP-1-associated histone H3, lysine 9- in Saccharomyces cerevisiae is linked to transcriptional elongation by specific methyltransferase that contributes to HP1-mediated silenc- RNA polymerase II. Mol Cell Biol 23:4207–4218 ing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 87. Li B, Howe L, Anderson S, Yates III JR, Workman JL 2003 The Set2 16:919–932. histone methyltransferase functions through the phosphorylated 107. Gyory I, Wu J, Fejer G, Seto E, Wright KL 2004 PRDI-BF1 recruits carboxyl-terminal domain of RNA polymerase II. J Biol Chem 278: the histone H3 methyltransferase G9a in transcriptional silencing. 8897–8903 Nat Immunol 5:299–308 88. Landry J, Sutton A, Hesman T, Min J, Xu RM, Johnston M, 108. Yang L, Xia L, Wu DY, Wang H, Chansky HA, Schubach WH, Sternglanz R 2003 Set2-catalyzed methylation of histone H3 re- Hickstein DD, Zhang Y 2002 Molecular cloning of ESET, a novel 168 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

histone H3-specific methyltransferase that interacts with ERG tran- Morillon A, Weise C, Schreiber SL, Mellor J, Kouzarides T 2003 scription factor. Oncogene 21:148–152 Methylation of histone H3 K4 mediates association of the Isw1p 109. Wang H, An W, Cao R, Xia L, Erdjument-Bromage H, Chatton B, ATPase with chromatin. Mol Cell 12:1325–1332 Tempst P, Roeder RG, Zhang Y 2003 mAM facilitates conversion 130. Kouskouti A, Scheer E, Staub A, Tora L, Talianidis I 2004 Gene- by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause specific modulation of TAF10 function by SET9-mediated methyl- transcriptional repression. Mol Cell 12:475–487 ation. Mol Cell 14:175–182 110. Beisel C, Imhof A, Greene J, Kremmer E, Sauer F 2002 Histone 131. Gary JD, Clarke S 1998 RNA and protein interactions modulated methylation by the Drosophila epigenetic transcriptional regulator by protein arginine methylation. Prog Nucleic Acids Res Mol Biol Ash1. Nature 419:857–862 61:65–131 111. Byrd KN, Shearn A 2003 ASH1, a Drosophila trithorax group pro- 132. McBride AE, Silver PA 2001 State of the Arg: protein methylation tein, is required for methylation of lysine 4 residues on histone H3. at arginine comes of age. Cell 106:5–8 Proc Natl Acad Sci USA 100:11535–11540 133. Zhang X, Cheng XD 2003 Structure of the predominant protein 112. Kellum R 2003 Is HP1 an RNA detector that functions both in arginine methyltransferase PRMT1 and analysis of its binding to repression and activation? J Cell Biol 161:671–672 substrate peptides. Structure 11:509–520 113. Piacentini L, Fanti L, Berloco M, Perrini B, Pimpinelli S 2003 134. Miranda TB, Miranda M, Frankel A, Clarke S 2004 PRMT7 is a Heterochromatin protein 1 (HP1) is associated with induced gene member of the protein arginine methyltransferase family with a expression in Drosophila euchromatin. J Cell Biol 161:707–714 distinct substrate specificity. J Biol Chem 279:22902–22907 114. Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, 135. Paik WK, Kim S 1967 Enzymatic methylation of protein fractions Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y 2003 Role from calf thymus nuclei. Biochem Biophys Res Commun 29:14–20 of histone H3 lysine 27 methylation in X inactivation. Science 300: 136. Paik WK, Kim S 1968 Protein methylase I. Purification and prop- 131–135 erties of the enzyme. J Biol Chem 243:2108–2114 115. Silva J, Mak W, Zvetkova I, Appanah R, Nesterova TB, Webster 137. Henry MF, Silver PA 1996 A novel methyltransferase (Hmt1p) Z, Peters AH, Jenuwein T, Otte AP, Brockdorff N 2003 Establish- modifies poly(A)ϩ-RNA-binding proteins. Mol Cell Biol 16:3668– ment of histone H3 methylation on the inactive X chromosome 3678 requires transient recruitment of Eed-Enx1 polycomb group com- 138. Gary JD, Lin WJ, Yang MC, Herschman HR, Clarke S 1996 The plexes. Dev Cell 4:481–495 predominant protein-arginine methyltransferase from Saccharomy- 116. Boggs BA, Cheung P, Heard E, Spector DL, Chinault AC, Allis CD ces cerevisiae. J Biol Chem 271:12585–12594 2002 Differentially methylated forms of histone H3 show unique 139. Lin W-J, Gary JD, Yang MC, Clarke S, Herschman HR 1996 The association patterns with inactive human X chromosomes. Nat mammalian immediate-early TIS21 protein and the leukemia- Genet 30:73–76 associated BTG1 protein interact with a protein-arginine N-meth- 117. Peters AH, Mermoud JE, O’Carroll D, Pagani M, Schweizer D, yltransferase. J Biol Chem 271:15034–15044 Brockdorff N, Jenuwein T 2002 Histone H3 lysine 9 methylation 140. Abramovich C, Yakobson B, Chebath J, Revel M 1997 A protein- is an epigenetic imprint of facultative heterochromatin. Nat Genet arginine methyltransferase binds to the intracytoplasmic domain of 30:77–80 the IFNAR1 chain in the type I interferon receptor. EMBO J 16: 118. Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, Spector DL 260–266 2001 Methylation of histone H3 at Lys-9 is an early mark on the X 141. Scott HS, Antonarakis SE, Lalioti MD, Rossier C, Silver PA, chromosome during X inactivation. Cell 107:727–738 Henry MF 1998 Identification and characterization of two putative 119. Czermin B, Melfi R, McCabe D, Seitz V, Imhof A, Pirrotta V 2002 human arginine methyltransferases (HRMT1L1 and HRMT1L2). Drosophila enhancer of Zeste/ESC complexes have a histone H3 Genomics 48:330–340 methyltransferase activity that marks chromosomal polycomb 142. Tang J, Gary JD, Clarke S, Herschman HR 1998 PRMT3, a type I sites. Cell 111:185–196 120. Muller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, Wild B, protein arginine N-methyltransferase that differs from PRMT1 in Miller EL, O’Connor MB, Kingston RE, Simon JA 2002 Histone its oligomerization, subcellular localization, substrate specificity, methyltransferase activity of a Drosophila polycomb group repres- and regulation. J Biol Chem 273:16935–16945 sor complex. Cell 111:197–208 143. Pollack BP, Kotenko SV, He W, Izotova LS, Barnoski BL, Pestka 121. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, S 1999 The human homologue of the yeast proteins Skb1 and Hsl7p Reinberg D 2002 Histone methyltransferase activity associated interacts with Jak kinases and contains protein methyltransferase with a human multiprotein complex containing the enhancer of activity. J Biol Chem 274:31531–31542 zeste protein. Genes Dev 16:2893–2905 144. Rho J, Choi S, Seong YR, Cho WK, Kim SH, Im DS 2001 Prmt5, 122. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst which forms distinct homo-oligomers, is a member of the protein- P, Jones RS, Zhang Y 2002 Role of histone H3 lysine 27 methylation arginine methyltransferase family. J Biol Chem 276:11393–11401 in polycomb-group silencing. Science 298:1039–1043 145. Frankel A, Yadav N, Lee J, Branscombe TL, Clarke S, Bedford MT 123. Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, Khorasanizadeh 2002 The novel human protein arginine N-methyltransferase S 2003 Molecular basis for the discrimination of repressive methyl- PRMT6 is a nuclear enzyme displaying unique substrate specificity. lysine marks in histone H3 by polycomb and HP1 chromodomains. J Biol Chem 277:3537–3543 Genes Dev 17:1870–1881 146. Najbauer J, Johnson BA, Young AL, Aswad DW 1993 Peptides 124. Min J, Zhang Y, Xu RM 2003 Structural basis for specific binding with sequences similar to glycine, arginine-rich motifs in proteins of polycomb chromodomain to histone H3 methylated at Lys 27. interacting with RNA are efficiently recognized by methyltrans- Genes Dev 17:1823–1828 ferase(s) modifying arginine in numerous proteins. J Biol Chem 125. Simon JA, Tamkun JW 2002 Programming off and on states in 268:10501–10509 chromatin: mechanisms of polycomb and trithorax group com- 147. Smith JJ, Ru¨ cknagel KP, Schierhorn A, Tang J, Nemeth A, Linder plexes. Curr Opin Genet Dev 12:210–218 M, Herschman HR, Wahle E 1999 Unusual sites of arginine meth- 126. Horard B, Tatout C, Poux S, Pirrotta V 2000 Structure of a poly- ylation in poly(A)-binding protein II and in vitro methylation by comb response element and in vitro binding of polycomb group protein arginine methyltransferases PRMT1 and PRMT3. J Biol complexes containing GAGA factor. Mol Cell Biol 20:3187–3197 Chem 274:13229–13234 127. Brown JL, Mucci D, Whiteley M, Dirksen ML, Kassis JA 1998 The 148. Li H, Park S, Kilburn B, Jelinek MA, Henschen-Edman A, Aswad Drosophila polycomb group gene pleiohomeotic encodes a DNA DW, Stallcup MR, Laird-Offringa IA 2002 Lipopolysaccharide- binding protein with homology to the transcription factor YY1. Mol induced methylation of HuR, an mRNA-stabilizing protein, by Cell 1:1057–1064 CARM1. J Biol Chem 277:44623–44630 128. Mulholland NM, King IF, Kingston RE 2003 Regulation of Poly- 149. Weiss VH, McBride AE, Soriano MA, Filman DJ, Silver PA, Hogle comb group complexes by the sequence-specific DNA binding JM 2000 The structure and oligomerization of the yeast arginine proteins zeste and GAGA. Genes Dev 17:2741–2746 methyltransferase, Hmt1. Nat Struct Biol 7:1165–1171 129. Santos-Rosa H, Schneider R, Bernstein BE, Karabetsou N, 150. Zhang X, Zhou L, Cheng X 2000 Crystal structure of the conserved Lee et al. • Protein Methylation and Transcription Endocrine Reviews, April 2005, 26(2):147–170 169

core of protein arginine methyltransferase PRMT3. EMBO J 19: 172. An W, Kim J, Roeder RG 2004 Ordered cooperative functions of 3509–3519 PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell 151. Frankel A, Clarke S 2000 PRMT3 is a distinct member of the protein 117:735–748 arginine N-methyltransferase family. Conferral of substrate spec- 173. Daujat S, Bauer UM, Shah V, Turner B, Berger S, Kouzarides T ificity by a zinc-finger domain. J Biol Chem 275:32974–32982 2002 Crosstalk between CARM1 methylation and CBP acetylation 152. Teyssier C, Chen D, Stallcup MR 2002 Requirement for multiple on histone H3. Curr Biol 12:2090–2097 domains of the protein arginine methyltransferase CARM1 in 174. Pal S, Yun R, Datta A, Lacomis L, Erdjument-Bromage H, Kumar its transcriptional coactivator function. J Biol Chem 277: J, Tempst P, Sif S 2003 mSin3A/histone deacetylase 2- and PRMT5- 46066–46072 containing Brg1 complex is involved in transcriptional repression 153. Hermanson O, Glass CK, Rosenfeld MG 2002 Nuclear receptor of the Myc target gene cad. Mol Cell Biol 23:7475–7487 coregulators: multiple modes of modification. Trends Endocrinol 175. Koh SS, Li H, Lee YH, Widelitz RB, Chuong CM, Stallcup MR Metab 13:55–60 2002 Synergistic coactivator function by coactivator-associated ar- 154. Beato M, Herrlich P, Schu¨tz G 1995 Steroid hormone receptors: ginine methyltransferase (CARM) 1 and ␤-catenin with two dif- many actors in search of a plot. Cell 83:851–857 ferent classes of DNA-binding transcriptional activators. J Biol 155. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and or- Chem 277:26031–26035 phan receptors. Cell 83:841–850 176. Rezai-Zadeh N, Zhang X, Namour F, Fejer G, Wen YD, Yao YL, 156. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene Gyory I, Wright K, Seto E 2003 Targeted recruitment of a histone expression by nuclear receptors and coregulators. Cell 108:465–474 H4-specific methyltransferase by the transcription factor YY1. 157. Glass CK, Rosenfeld MG 2000 The coregulator exchange in Genes Dev 17:1019–1029 transcriptional functions of nuclear receptors. Genes Dev 14: 177. Xu W, Cho H, Kadam S, Banayo EM, Anderson S, Yates III JR, 121–141 Emerson BM, Evans RM 2004 A methylation-mediator complex in 158. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, hormone signaling. Genes Dev 18:144–156 Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor co- 178. Lee YH, Campbell HD, Stallcup MR 2004 Developmentally es- activator ACTR is a novel histone acetyltransferase and forms a sential protein flightless I is a nuclear receptor coactivator with multimeric activation complex with P/CAF and CBP/p300. Cell actin binding activity. Mol Cell Biol 24:2103–2117 90:569–580 179. Fabbrizio E, El Messaoudi S, Polanowska J, Paul C, Cook JR, Lee 159. Voegel JJ, Heine MJS, Tini M, Vivat V, Chambon P, Gronemeyer JH, Negre V, Rousset M, Pestka S, Le Cam A, Sardet C 2002 H 1998 The coactivator TIF2 contains three nuclear receptor bind- Negative regulation of transcription by the type II arginine meth- ing motifs and mediates transactivation through CBP binding- yltransferase PRMT5. EMBO Rep 3:641–645 dependent and -independent pathways. EMBO J 17:507–519 180. Rogatsky I, Zarember KA, Yamamoto KR 2001 Factor recruitment 160. Koh SS, Chen D, Lee Y-H, Stallcup MR 2001 Synergistic enhance- and TIF2/GRIP1 corepressor activity at a collagenase-3 response ment of nuclear receptor function by p160 coactivators and two element that mediates regulation by phorbol esters and hormones. coactivators with protein methyltransferase activities. J Biol Chem EMBO J 20:6071–6083 276:1089–1098 181. Vo N, Goodman RH 2001 CREB-binding protein and p300 in 161. Schurter BT, Koh SS, Chen D, Bunick GJ, Harp JM, Hanson L, transcriptional regulation. J Biol Chem 276:13505–13508 Henschen-Edman A, Mackay DR, Stallcup MR, Aswad DW 2001 182. Chevillard-Briet M, Trouche D, Vandel L 2002 Control of CBP Methylation of histone H3 by coactivator-associated arginine meth- co-activating activity by arginine methylation. EMBO J 21:5457– yltransferase 1. Biochem 40:5747–5756 5466 162. Chen D, Huang S-M, Stallcup MR 2000 Synergistic, p160 coacti- 183. Mowen KA, Tang J, Zhu W, Schurter BT, Shuai K, Herschman vator-dependent enhancement of estrogen receptor function by HR, David M 2001 Arginine methylation of STAT1 modulates CARM1 and p300. J Biol Chem 275:40810–40816 IFN␣/␤-induced transcription. Cell 104:731–741 163. Lee Y-H, Koh SS, Zhang X, Cheng X, Stallcup MR 2002 Synergy 184. Kwak YT, Guo J, Prajapati S, Park KJ, Surabhi RM, Miller B, among nuclear receptor coactivators: selective requirement for pro- Gehrig P, Gaynor RB 2003 Methylation of SPT5 regulates its in- tein methyltransferase and acetyltransferase activities. Mol Cell teraction with RNA polymerase II and transcriptional elongation Biol 22:3621–3632 properties. Mol Cell 11:1055–1066 164. Ma H, Baumann CT, Li H, Strahl BD, Rice R, Jelinek MA, Aswad 185. Friesen WJ, Massenet S, Paushkin S, Wyce A, Dreyfuss G 2001 DW, Allis CD, Hager GL, Stallcup MR 2001 Hormone-dependent, SMN, the product of the spinal muscular atrophy gene, binds CARM1-directed, arginine-specific methylation of histone H3 on a preferentially to dimethylarginine-containing protein targets. Mol steroid-regulated promoter. Curr Biol 11:1981–1985 Cell 7:1111–1117 165. Bauer UM, Daujat S, Nielsen SJ, Nightingale K, Kouzarides T 186. Meister G, Eggert C, Buhler D, Brahms H, Kambach C, Fischer U 2002 Methylation at arginine 17 of histone H3 is linked to gene 2001 Methylation of Sm proteins by a complex containing PRMT5 activation. EMBO Rep 3:39–44 and the putative U snRNP assembly factor pICln. Curr Biol 11: 166. Strahl BD, Briggs SD, Brame CJ, Caldwell JA, Koh SS, Ma H, 1990–1994 Cook RG, Shabanowitz J, Hunt DF, Stallcup MR, Allis CD 2001 187. Liu Q, Dreyfuss G 1995 In vivo and in vitro arginine methylation Methylation of histone H4 at arginine 3 occurs in vivo and is of RNA-binding proteins. Mol Cell Biol 15:2800–2808 mediated by the nuclear receptor coactivator PRMT1. Curr Biol 188. Green DM, Marfatia KA, Crafton EB, Zhang X, Cheng X, Corbett 11:996–1000 AH 2002 Nab2p is required for poly(A) RNA export in Saccharo- 167. Wang H, Huang Z-Q, Xia L, Feng Q, Erdjument-Bromage H, myces cerevisiae and is regulated by arginine methylation via Strahl BD, Briggs SD, Allis CD, Wong J, Tempst P, Zhang Y 2001 Hmt1p. J Biol Chem 277:7752–7760 Methylation of histone H4 at arginine 3 facilitates transcriptional 189. Lee J, Bedford MT 2002 PABP1 identified as an arginine methyl- activation by nuclear hormone receptor. Science 293:853–857 transferase substrate using high-density protein arrays. EMBO Rep 168. Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, 3:268–273 Gannon F 2003 Estrogen receptor-␣ directs ordered, cyclical, and 190. Nichols RC, Wang XW, Tang J, Hamilton BJ, High FA, Hersch- combinatorial recruitment of cofactors on a natural target pro- man HR, Rigby WF 2000 The RGG domain in hnRNP A2 affects moter. Cell 115:751–763 subcellular localization. Exp Cell Res 256:522–532 169. Qi C, Chang J, Zhu Y, Yeldandi AV, Rao SM, Zhu YJ 2002 Iden- 191. Cote J, Boisvert FM, Boulanger MC, Bedford MT, Richard S 2003 tification of protein arginine methyltransferase 2 as a coactivator for Sam68 RNA binding protein is an in vivo substrate for protein estrogen receptor ␣. J Biol Chem 277:28624–28630 arginine N-methyltransferase 1. Mol Biol Cell 14:274–287 170. Rice JC, Allis CD 2001 Histone methylation versus histone acet- 192. Rajpurohit R, Paik WK, Kim S 1994 Effect of enzymic methylation: new insights into epigenetic regulation. Curr Opin Cell Biol ylation of heterogeneous ribonucleoprotein particle A1 on its 13:263–273 nucleic-acid binding and controlled proteolysis. Biochem J 171. Fischle W, Wang YM, Allis CD 2003 Histone and chromatin cross- 304(Pt 3):903–909 talk. Curr Opin Cell Biol 15:172–183 193. Rajpurohit R, Lee SO, Park JO, Paik WK, Kim S 1994 Enzymatic 170 Endocrine Reviews, April 2005, 26(2):147–170 Lee et al. • Protein Methylation and Transcription

methylation of recombinant heterogeneous nuclear RNP protein 200. Saccani S, Natoli G 2002 Dynamic changes in histone H3 Lys 9 A1. Dual substrate specificity for S-adenosylmethionine:histone- methylation occurring at tightly regulated inducible inflammatory arginine N-methyltransferase. J Biol Chem 269:1075–1082 genes. Genes Dev 16:2219–2224 194. Gallouzi IE, Brennan CM, Steitz JA 2001 Protein ligands mediate 201. Li J, Lin Q, Yoon HG, Huang ZQ, Strahl BD, Allis CD, Wong J the CRM1-dependent export of HuR in response to heat shock. 2002 Involvement of histone methylation and phosphorylation in RNA 7:1348–1361 regulation of transcription by thyroid hormone receptor. Mol Cell 195. Cimato TR, Tang J, Xu Y, Guarnaccia C, Herschman HR, Pongor Biol 22:5688–5697 S, Aletta JM 2002 Nerve growth factor-mediated increases in pro- 202. Rice JC, Nishioka K, Sarma K, Steward R, Reinberg D, Allis CD tein methylation occur predominantly at type I arginine methyl- 2002 Mitotic-specific methylation of histone H4 Lys 20 follows ation sites and involve protein arginine methyltransferase 1. J Neu- increased PR-Set7 expression and its localization to mitotic chro- rosci Res 67:435–442 mosomes. Genes Dev 16:2225–2230 196. Cimato TR, Ettinger MJ, Zhou X, Aletta JM 1997 Nerve growth 203. Kim J, Jia L, Tilley WD, Coetzee GA 2003 Dynamic methylation factor-specific regulation of protein methylation during neuronal of histone H3 at lysine 4 in transcriptional regulation by the an- differentiation of PC12 cells. J Cell Biol 138:1089–1103 drogen receptor. Nucleic Acids Res 31:6741–6747 197. Annunziato AT, Eason MB, Perry CA 1995 Relationship between 204. Allis CD, Bowen JK, Abraham GN, Glover CV, Gorovsky MA methylation and acetylation of arginine-rich histones in cycling and 1980 Proteolytic processing of histone H3 in chromatin: a phys- arrested HeLa cells. Biochem 34:2916–2924 iologically regulated event in Tetrahymena micronuclei. Cell 20: 198. Hendzel MJ, Davie JR 1991 Dynamically acetylated histones of 55–64 chicken erythrocytes are selectively methylated. Biochem J 273: 205. Ahmad K, Henikoff S 2002 The histone variant H3.3 marks active 753–758 chromatin by replication-independent nucleosome assembly. Mol 199. Desrosiers R, Tanguay RM 1988 Methylation of Drosophila his- Cell 9:1191–1200 tones at proline, lysine, and arginine residues during heat shock. 206. Bannister AJ, Schneider R, Kouzarides T 2002 Histone methyl- J Biol Chem 263:4686–4692 ation: dynamic or static? Cell 109:801–806

7th International Symposium on VIP, PACAP, and Related Peptides

The 7th International Symposium on VIP, PACAP, and related peptides will be held in Rouen, Normandy, France, September 11–14, 2005. The conference will present recent advances in the fields of physiology and pharmacology of VIP, PACAP, related peptides, and their receptors.

For information see: http://vip-pacap2005.crihan.fr.

For inquiries please contact: Hubert Vaudry, Inserm U413, at [email protected], or Marc Laburthe, Inserm U410, at [email protected].

Endocrine Reviews is published bimonthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.