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Posttranslational Modifications of Histones by Methylation
POSTTRANSLATIONAL MODIFICATIONS OF HISTONES BY METHYLATION By ADAM WOOD* AND ALI SHILATIFARDÀ1
*Department of Biochemistry, Saint Louis University School of Medicine, À St. Louis, Missouri; The Saint Louis University Cancer Center, Saint Louis University School of Medicine, St. Louis, Missouri
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . II. Lysine Methyltransferases . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . A. SET Domain Containing Lysine-Specific Histone Methyltransferases . .. . . . . . B. Non-SET Domain Containing Lysine Methyltransferases. . . . . . . . . . . . . . . . .. . . . . . III. Arginine Methyltransferases . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . A. Structure . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . B. Catalytic Mechanism. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . C. Identified Arginine Target Residues . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . IV. Epilogue . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .
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I. Introduction During development, cells become committed to different fates, in part through heritable, quasi-stable changes in gene expression. The mechanisms by which the 2-m-long DNA of eukaryotic organisms is packaged into the cell nucleus while remaining functional still remains not well understood. However, the last few years have been a watershed for understanding the first stage in this packaging process: the formation of the nucleosome core particle. Nucleosomes were first observed by viewing electron micrographs of lysed nuclei (Kornberg, 1974). The chromatin appears as a series of ‘‘beads on a string,’’ the beads being the individual nucleosomes and the ‘‘string’’ the linker DNA. Since the discovery of the ‘‘beads on a string,’’ it has been demonstrated that each nucleosome consists of eight core histone proteins (two each of H3, H4, H2A, and H2B; Luger et al., 1997). Each of the core histones contains a shared region of homology termed the histone fold, and it is this motif that allows for histone–DNA contacts and for dimerization of histones (Arents and Moudrianakis, 1995). Two H3/H4 heterodimers interact via a four-helix bundle formed between the two H3 histone folds, and this tetramer is then
1 Correspondence to: Ali Shilatifard, Saint Louis University School of Medicine, Department of Biochemistry, 1402 South Grand Blvd. St. Louis, MO 63104 (e-mail: [email protected]).
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flanked by two H2B/H2A heterodimers with two four-helix bundles linking histones H2B and H4, thus bringing the core octamer together (Luger et al., 1997). The core histones are wrapped by 147 base pairs of DNA (1.65 turns around the histone octamer) in a left-handed superhelix, forming the intact nucleosome. The nucleosome structure is stabilized by about 142 hydrogen bonds between the DNA and the core histones, and several hydrophobic interactions are present between DNA and histones within the core (Luger et al., 1997). The core histones are rich in lysine and arginine residues, whose positive charge can form stable interactions with the negatively charged phosphodiester backbone of DNA. Taken together, these interactions allow for a highly stable complex to form between DNA and the core histone proteins. The nucleosome structure, when further stabilized by the linker histone H1, can compact the linear DNA about 30–40-fold, which is a significant reduction in length (Luger et al., 1997). This compaction affects the binding of nonhistone proteins, such as transcription factors, by restricting access to the binding sites within the DNA. Reduction in DNA length produced by histone-induced supercoiling in a nucleosome is significant and is an essential first step in the formation of higher-order chromatin structures. During the last decade, there has been an explosion of information regarding the role of nucleosomes in the regulation of gene expression (Workman and Kingston, 1998). Just as exciting has also been the growing possibility that the nucleosomes can transmit epigenetic information from one cell generation to the next (Jenuwein and Allis, 2001; Turner, 2002). The histone amino termini, or tails, are the sites of many of the covalent modifications that alter the nucleosome structure (Fig. 1). These tails extend away from the core of the nucleosome and are available for interactions with the DNA or with other proteins (Luger et al., 1997). There are multiple modification sites on each histone tail, and some amino acids in the histone tail can be modified in two or more ways (Schreiber and Bernstein, 2002; Turner, 2002; Figs. 1, 2). Because the sequence of the N-terminal tails is rich in arginine and lysine residues, the histone tails are very basic; covalent modification of these histone residues such as acetylation can alter the charge and structural properties of the nucleosome and can serve to restrict or allow access to the nucleosomal DNA. Recently, it has been demonstrated that modifications can also occur within the globular domain of histone H3. Gottschling and coworkers and Struhl and colleagues independently demonstrated that methylation on lysine 79 of histone H3, which occurs within the globular domain, restricts the binding of silencing proteins such as Sir2 to specific regions of the genome (Ng et al., 2002a; Van Leeuwen et al., 2002). The deletion of Dot1, which
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Fig. 1. Nucleosome structure and histone tail modification sites.
result in the loss of histone H3 methylation on lysine 79, as will be discussed later, abolishes the interaction of the silencing proteins in the body of genes and causes silencing defects at the telomers (Ng et al., 2002a; Van Leewen et al., 2002). Several known covalent modifications have been observed so far that can modify the amino acid residues in the histone tails. There include acetylation, phosphorylation, ubiquitination, and methylation. Although some of these modifications have been known for many years, only recently have functional roles for these modifications begun to surface (Workman and Kingston, 1998). Each histone can undergo numerous modifications, and the combinatorial effect of these serves to elicit a multitude of different responses. This combinatorial modification of histone tails has been referred to as the ‘‘histone code’’ (Jenuwein and Allis, 2001; Strahl and Allis, 2000) and has been proposed to play a pivotal role in the regulation of gene expression (Fig. 3). Histone acetylation was one of the first posttranslational histone proteins to be described and demonstrated to function in the regulation of transcriptional activation (Braunstein et al., 1993). To date, this modification of histone is the one best characterized (Workman and Kingston, 1998). Acetylation of a lysine residue on a histone tail can neutralize the positive charge, thus weakening the interaction of the nucleosome with the DNA backbone. This then allows for the ‘‘remodeling’’ of the nucleosome so that the transcriptional machinery, as well as other proteins, can gain access to previously restricted sites within the DNA
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Fig. 2. Histone residues modified by methylation and the enzymes responsible for each.
(Kristjuhan et al., 2002). The acetylation of histone tail has come to be known as a hallmark of gene-specific transcriptional activation, and conversely, histone deacetylation, catalyzed by HDACs, play a pivotal role in transcriptional repression (Strahl and Allis, 2000; Utley et al., 1998; Wolffe, 2001; Zhou et al., 2002). HDACs are also found in large protein complexes that function specifically at promoters. Phosphorylation of histone tails has recently been discovered as another covalent modification of histones. The covalent modification of histones by phosphorylation has been demonstrated to be involved in DNA repair, chromosomal condensation, apoptotic signaling, and heat shock–induced pathways (Ballal et al., 1975; Berger, 2001; Hunt and Dayhoff, 1977; Nowak and Corces, 2000; Rogakou et al., 2000; Van Hooser et al., 1998). Interestingly, phosphorylation of serine 10 of histone H3, catalyzed by the protein kinases Ipl1, Rsk2, and Msk1 (Berger, 2001), also alter chromatin structure and function and affect transcriptional activation, condensation of chromosomes during mitosis and meiosis, and regulation of cell division.
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Fig. 3. General information for known SET domain and PRMT proteins (* undefined).
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Histone ubiquitination was discovered as a modification of histone H2A more than 20 years ago (Ballal et al., 1975; Hunt and Dayhoff, 1977). Only recently, though, has a functional role for the ubiquitination of histone proteins been described. In Saccharomyces cerevisiae, it has been demonstrated that Rad6 is involved in the attachment of ubiquitin to lysine 123 of histone H2B (Robzyk et al., 2000). This modification of histone H2B is required as a signal for histone methylation on lysines 4 and 79 of histone H3 (Dover et al., 2002; Ng et al., 2002a, 2002b; Sun and Allis, 2002). Rad6 has been demonstrated to be involved in several different pathways such as DNA repair (Jentsch et al., 1987), proteolytic degradation (Dohmen et al., 1991), and transcriptional silencing (Huang et al., 1997). Therefore, Rad6 requires an specific E3 ligase to direct it only to its role in transcription (Wood et al., 2003). Several lines of evidence have demonstrated that Bre1 is the E3 ligase for Rad6 in transcription. First, Bre1 is found in a macromolecular complex with Rad6; second, Bre1 is required for the ubiquitination of histone H2B in vivo, which seems to be a signal for methylation of histone H3 at its 4th and 79th lysines; third, Bre1 is required for telomeric silencing; fourth, Bre1 is recruited with Rad6 to a promoter; fifth, Bre1 is essential for the recruitment of Rad6 to chromatin at a promoter; and sixth, Bre1 is dedicated to the transcriptional regulatory role of Rad6. Much like histone ubiquitination, histone methylation was discovered several decades ago, but the biological significance of this modification remained elusive. However, its functional role in the regulation of gene expression is now becoming clearer. The attachment of methyl groups to histone proteins occurs predominantly at specific lysine or arginine residues on histones H3 and H4 (Jenuwein and Allis, 2001; Schreiber and Bernstein, 2002; Stallcup, 2001; Turner, 2002; Figs. 1, 2). This modification, unlike acetylation, phosphorylation, and ubiquitination, is stable. Although deacetylases, phosphatases, and deubiquitinating enzymes have all been described, as of yet there is no known demethylase that can function on methylated histone. We have recently demonstrated that an RNA polymerase II elongation factor, the Paf1 complex, is required for the recruitment of histone methyltransferases to the elongating RNA polymerase, and have therefore suggested that it is possible that modification by methylation may serve as a mark of transcriptional memory for transcribed genes. To date, two types of histone methylation have been observed: methylation of the "-amino group of lysine, and methylation of the N nitrogens of the arginine side chain guanido group (Aletta et al., 1998; Rea et al., 2000).
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II. Lysine Methyltransferases A. SET Domain Containing Lysine-Specific Histone Methyltransferases The first histone methyltransferase to be identified was the product of the Drosophila suppressor of position-effect variegation gene, Su(var)3-9, Clr4+ (cryptic locus regulator) in Schizosaccharomyces pombe, and SUV39H1 and SUV39H2 in human (Rea et al., 2000). Su(var)3-9 contains a SET domain, a highly conserved motif originally identified at the C-terminal ends of Su(var)3-9, enhancer of zeste (a member of the Polycomb family), and Trithorax, three gene-regulatory factors in Drosophila (Dorn et al., 1993; Jones and Gelbart, 1993; Tschiersch et al., 1994; reviewed in Alvarez-Venegas and Avramova, 2002). In yeast, the product of the SET1 gene is found in a macromolecular complex called COMPASS (complex of proteins associated with Set1; Miller et al., 2001; Nagy et al., 2002; Roguev et al., 2001). COMPASS can methylate the fourth lysine of histone H3 and thereby effect regulation of transcription of genes located near chromosome telomeres. To date, COMPASS is the only histone methyl transferase that was biochemically isolated and that functions in a macromolecular complex (Krogan et al., 2002). The yeast Set1 protein alone is not capable of methylating histone H3 and requires the presence of other components of COMPASS for its enzymatic function. It has been proposed that components of COMPASS may function either in properly positioning the catalytic domain of Set1 or in proper substrate recognition by Set1 (Krogan et al., 2002). On the basis of homology of the conserved SET domains, this family of proteins is classified into four families: Su(var)3-9, E(z), TRX, and ASH1 (Alvarez-Venegas and Avramova, 2002; Jenuwein et al., 1998). Since its initial discovery, the SET domain has been described as part of several proteins involved in modulating gene activity in eukaryotes ranging from yeast to humans (Alvarez-Venegas and Avramova, 2002; Jenuwein et al., 1998; Nislow et al., 1997). This modulation happens via the histone methyltransferase activity found in many SET domain proteins and the complexes containing them. These SET domain-containing protein complexes have a number of identified lysine residue targets, and as we will describe later, the methylation of each different residue can elicit an array of different cellular responses. Because of their fairly recent discovery, only a few SET domain-containing enzymes and their target residues have been identified and crystallized (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). To date, all of the characterized SET containing proteins have been
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demonstrated to have a highly conserved active site and a binding site for S-adenosyl-L-methionine (SAM domain).
1. Structure The histone methltransferase activity (HMTase) of the SET-domaincontaining protein resides within the SET domain. The SET domain itself is approximately 130 amino acids in length (Fig. 4; Jenuwein et al., 1998). Although some areas of the sequence are highly conserved between species, others are much more varied. The pre-SET and post-SET domains can differ in sequence and structure between different HMTases. However, members of the same SET domain protein families have high
Fig. 4. Sequence alignment of some known lysine methyltransferases. The residues involved in the catalytic domain ‘‘knot’’ are indicated by red bars. The conserved active site tyrosine residue is denoted by *.
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homology in these pre-SET and post-SET domains. In each case, however, the pre-SET and post-SET domains are required for methyltransferase activity (Jacobs et al., 2002; Min et al., 2002; Rea et al., 2000; Wilson et al., 2002; Zhang et al., 2002). In many cases, these pre-SET and post-SET domains intertwine with the SET domain itself to alter the tertiary structure of the enzymes (Min et al., 2002). These variations lend different site specificities to each enzyme for methyl group attachment. However, the catalytic core of the enzyme and the SAM substrate binding site remain highly conserved. In the Su(var)3-9 and E(z) families of HMTases, the pre-SET region is rich in cysteine residues that form a triangular zinc cluster that acts to bind zinc ions very tightly, an interaction that serves to stabilize the structure (Min et al., 2002). This stabilization and positioning of the zinc cluster is essential for activity, as mutations in conserved residues that serve to position the cluster result in an inactive enzyme (Min et al., 2002). Enzymes containing this zinc cluster are also sensitive to metal chelating agents such as EDTA (Min et al., 2002; Zhang et al., 2002). Although some TRX family members also contain cysteine-rich regions N-terminal to the SET domain, these regions are at a considerable distance from the SET domain itself and may serve other functions. The members of the Su(var)3-9 family of HMTs are the only SET-domain-containing enzymes shown to have a cysteine-rich post-SET domain. Apart from the zinc clusters, many of the identified HMTases have similarities in the region N-terminal to the SET domain. The crystal structure of SET7/9 reveals the presence of an acidic groove on the pre-SET domain surface of the protein, which extends to the SET domain itself (Wilson et al., 2002). This groove may serve as the binding site for the basic N-terminal tails of histone proteins, and mutations of residues within the groove result in aberrant substrate binding. The place in which pre-SET groove intersects the SET domain also happens to be where the adenine moiety of AdoMet binds (Wilson et al., 2002). However, it should be noted that several other grooves on the surface of the SET domain surface approach the AdoMet binding site and that, as of yet, it is unclear which is responsible for histone tail binding. Although the adenine ring of AdoMet makes several contacts with the enzyme, it appears that the sulfonium component does not interact with the binding site (Wilson et al., 2002). However, the ribose ring forms Van der Waals interactions with a conserved residue in the core of the SET domain, as will be discussed later. The rest of the AdoMet binding site is a region of high sequence conservation, and the contacts between the site residues and AdoMet rely solely on hydrogen bonding. In fact, the amine nitrogen of AdoMet is hydrogen bonded to a highly conserved histidine residue on the ‘‘loop’’ of the active site knot (discussed later), and mutation of this residue abolishes enzyme activity (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002).
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The catalytic core of the SET domain is rich in -strands that comprise several regions of -sheets. The actual number of -strands may vary between different enzymes (eight and nine in Clr4 and SET7/9, respectively), but the number of alpha helices remains constant at two. In many cases, -strands found in the pre-SET region will form -sheets with the -strands of the SET domain, imparting slight variations to the SET domain structure. These small changes alter the target residue site specificity for methylation and allow the SET domain methyltransferases to target many different residues. This interplay between the pre-SET domain and the catalytic core is critical for enzyme function, and it has been demonstrated that both the pre-SET and post-SET regions that flank the SET domain are required for histone methyltransferase activity. The C-terminus region of the SET domain makes an unusual turn that threads back through a loop formed between a -strand and an alpha helix at the core of the domain to form a ‘‘knot’’ (Jacobs et al., 2002). This knot structure is the catalytic core of the enzyme. Conserved residues in the loop and C-terminal ‘‘thread’’ combine to establish a hydrophobic core in the knot, which serves to stabilize the structure. Further reinforcement of the active site structure is achieved by hydrogen bonding between several conserved residues within the knot (Fig. 4; Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). Both the hydrophobic core of the active site and the residues involved in the hydrogen bonding constitute regions of high sequence conservation. This ‘‘knotted’’ catalytic core is unique to SET domain containing enzymes and does not resemble any other previously characterized methyltransferases that use AdoMet as a methyl group donor. Within this core, AdoMet and the substrate lysine are brought into close proximity. The core of each SET domain protein contains a completely conserved tyrosine residue, which is critical to the active site function. The conserved tyrosine forms Van der Waals interactions with the ribose of AdoMet and also serves to deprotonate the "-amino group of the target lysine residue (Jacobs et al., 2002). This is critical for activity, as mutation of the tyrosine results in an inactive enzyme (Wilson et al., 2002). Note once again that the conserved residues that are critical for the catalytic ability of the SET domain are derived from the knot itself; thus, the knot structure is crucial for the formation of the active site and the activity of the SET domain itself.
2. Catalytic Mechanism of the SET Domain When AdoMet and the lysine of the substrate histone tail are bound and properly oriented in the catalytic pocket of the SET domain, the conserved tyrosine residue acts to deprotonate the "-amino group of the lysine
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Fig. 5. Catalytic data of characterized histone methyltransferases (* undefined value).
residue (Trievel et al., 2002; Fig. 6). This primes the lysine side chain to make a nucleophilic attack on the methyl group of the AdoMet molecule. The methyl group is transferred to the lysine side chain, resulting in the formation of methylated histone and the byproduct of AdoMet demethylation, AdoHcy (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). In vitro catalytic data have been obtained for only a few of the characterized SET domain containing proteins. These data are summarized in Fig. 5. Most of the examined HKMTs have optimal methyltransferase activity between a pH of 8 and 10. The mechanism for methyl group attachment to lysine residues is illustrated in Fig. 6.
3. Identified Lysine Target Residues To date, the lysine residue methylation of histone proteins by SETdomain-containing enzymes occurs specifically at Lys4, Lys9, Lys27, and Lys36 of histone H3, and Lys20 of H4, and each lysine residue can be methylated up to three times. As more SET-domain proteins are characterized, it is possible that more histone and nonhistone targets will be found as well. For the residues that have been established as targets for KHMTs, though, a number of biological consequences for their methylation such as transcriptional silencing and activation, have been observed (Fig. 3). What is interesting about some of these methyltransferases is that they are capable of methylating the same lysine residue once, twice, or three times. Studies performed in Kouzarides’s laboratory have demonstrated that dimethylated K4 of histone H3 is associated with active euchromatic regions, but not in silent heterochromatic sites (Santos-Rosa et al., 2002). They have also demonstrated that Set1 subunit of COMPASS can catalyze dimethylation and trimethylation of K4. This correlates with the activity of many genes. They have also developed antibodies that discriminate between the di- and trimethylated state of lysine 4 of histone H3 and have demonstrated that dimethylation occurs at both inactive and active euchromatic genes, whereas trimethylation is present exclusively at active genes.
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Fig. 6. Mechanism of methyl group transfer to lysine residues.
What remains to be determined is how COMPASS dimethylates at some sites and trimethylates at other sites. It is possible that either the subunits of COMPASS or other proteins within the yeast genome can participate in this process. Lysine 9 of histone H3 is the target of many SET-domain-containing enzymes (Fig. 2). Most of the HKMTs that methylate this residue belong to the Su(var)3-9 family. In Drosophila and humans, methylation of this lysine provides a binding site for the chromo domain of heterochromatin protein 1 (HP1; Aagaard et al., 2000; Loyola et al., 2001). Work done in Drosophila has demonstrated that the interaction of HP1 and methylated
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H3 K9 is localized to regions of heterochromatin on polytene chromosomes, and alteration of HP1 recruitment affects heterochromatic gene silencing (Jacobs et al., 2001). Methylation of H3 lysine 9 then may serve to mediate heterochromatin assembly and gene silencing through its recruitment of HP1 (Hall et al., 2002; Schotta et al., 2002). The methylation of lysine 4 of histone H3 has also been extensively studied. Although the methylation of lysine 9 in higher eukaryotes is strongly related to transcriptional repression, methylation of histone H3 lysine 4 seems to antagonize this effect. In mammals, SET7/9 specifically methylates lysine 4, and this methylation coincides with transcriptional activation (Nishioka et al., 2002). SET7/9 also lacks the cysteine-rich domains found in lysine 9-specific methyltransferases, once again demonstrating the role of the pre-SET and post-SET domains in substrate specificity (Rea et al., 2000; Wilson et al., 2002). In S. cerevisiae, the methylation of K4 is carried out by COMPASS, a large SET-domain-containing protein. In contrast to the Lys4 methylation in higher eukaryotes, SET1-dependent methylation is involved with transcriptional repression at the telomeres and with mating type and rDNA loci (Bryk et al., 2002; Krogan et al., 2002; Miller et al., 2001). Recently, it was demonstrated that methylation of lysine 79 of histone H3 can regulate telomere-associated gene silencing by restricting the binding of silencing proteins to the telomeres and other ‘‘silenced’’ regions of chromatin (Van Leeuwen et al., 2002). It is also very possible that methylation of histone H3 on lysine 4 may function via a similar mechanism.
B. Non-SET Domain Containing Lysine Methyltransferases A novel histone methyltransferase was recently described as having specificity for lysine 79 of histone H3 (Ng et al., 2002a; Van Leeuwen et al., 2002). This enzyme, named Dot1 (disruptor of telomeric silencing 1) was originally discovered based on a high–copy suppressor screen of telomeric silencing (Singer et al., 1998). It was recently demonstrated that the target—a lysine residue by Dot1—is not found in the tail region of the histone but, rather, in the globular core of the histone itself. The residues are located on the top and bottom of the nucleosome core (Van Leeuwen et al., 2002). To date, Dot1 is the only enzyme known to methylate residues within the histone core. In vitro assays have revealed another interesting characteristic of Dot1. Almost all other HMTs can methylate free histone, some even being able to methylate peptides having homology to their respective target sequence. Dot1, however, will only methylate if the nucleosome is intact. Most interestingly, it was recently demonstrated that
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histone methylation by COMPASS requires histone ubiquitination by Rad6 and its E3 ligase Bre1 (Ng et al., 2002a, 2002b; Wood et al., 2003). This modification of histone H2B by ubiquitination is also a required modification for Dot1’s function: linking the molecular function of a non-SET domain and a SET domain containing methyltransferase.
1. Structure Dot1, unlike other known lysine methyltransferases, does not contain a SET domain. Rather, the methyltransferase fold resembles those of known arginine methyltransferases. In fact, many of the conserved residues found in PRMTs that are important for methylation activity are found in Dot1, and like the other known HMTs, Dot1 uses AdoMet as a methyl donor. Very recently, the crystal structure of human Dot1 in complex with AdoMet was resolved (Min et al., 2003). The N terminus of hDot1 contains the active site, and the N-terminal and C-terminal domains of the Dot1 catalytic domain are linked by a loop that also serves as part of the AdoMet binding site. The loop, and thus the SAM binding site, is highly conserved between Dot1 and other AdoMet binding proteins. Mutations within this conserved domain disrupt AdoMet binding and methyltransferase activity. The SAM binding site also contains a channel positioned near the methyl group, possibly for the binding of substrate lysine residues. Mutations within this channel do in fact abolish methyltransferase activity without affecting the overall structure of the protein (Min et al., 2003). The structure of the C terminus of Dot1 was not resolved. However, because of its distance from the active site, it is believed to be important for the substrate specificity and binding of Dot1. This region carries a positive charge, which would allow for a favorable interaction with the negatively charged backbone of DNA (Min et al., 2003). This may also explain why Dot1 is only able to methylate histone H3 in the context of an intact nucleosome.
2. Identified Lysine Target Residues The methylation of H3 K79 catalyzed by Dot1 is important for silencing at the telomeres and is believed be involved in the recruitment of Sir2 and Sir4 to these regions of DNA. A proposed mechanism is that histone methylation by Dot1 restricts Sir protein binding to the telomeres, and loss or overexpression of Dot1 function allows for promiscuous binding of the Sir proteins to chromatin within the genome, effectively titrating them away from the telomeres (Ng et al., 2002a; Van Leeuwen et al., 2002). The loss of the Sir proteins from the telomeres confers silencing defects to Dot1 deficient strains, and thus, methylation of Lysine 79 serves to restrict silencing to specific regions of the genome.
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III. Arginine Methyltransferases The methylation of arginine residues of histone proteins has also received much attention in recent years. However, not much is known about the biological significance of this process as of yet. Recent experiments have identified three distinct types of methylation that occur at arginine residues on histone tails along with two types of PRMTs that accomplish this task. To date, the three known products of arginine methylation are NG-monomethylarginine, NGNG-symmetric dimethylarginine (in which both guanido nitrogens are methylated), or NGN’G-asymmetric dimethylarginine (in which only one guanido nitrogen receives two methyl groups; McBride and Silver, 2001; Stallcup, 2001). The two types of PRMTs are classified by the type of methylation they accomplish. Type I PRMTs (PRMT1, PRMT3, CARM1/PRMT4, and Rmt1/Hmt1) produce monomethylarginine and asymmetric dimethylarginine (Chen et al., 1999; Frankel and Clarke, 2000; Gary et al., 1996; McBride et al., 2000) whereas type II methyltransferases (JBP1/PRMT5), have the ability to form monomethyl or symmetric dimethylarginine (Branscombe et al., 2001). These differences arise from variations in the arginine binding pocket, which can accommodate a previously methylated guanido group (type II) and then allow the other to be modified (symmetric), or a pocket that only allows one of the guanido nitrogens to be modified (asymmetric type 1; Branscombe et al., 2001). Like the SET domain containing methyltransferases, PRMTs require AdoMet as a cofactor and methyl group donor. The PRMTs identified so far exist as members of multisubunit complexes, the formation of which is critical for in vivo methyltransferase activity (Teyssier et al., 2002). Many of the arginine methyltransferases also form homo-dimers or homooligomers, a step that is required for their catalytic activity (Weiss et al., 2000). This need to oligomerize has not been demonstrated for any known lysine methyltransferases. The identities of several protein arginine methyltransferases are now known, but only a few have been shown to have specificity for histone proteins. The mammalian PRMT1, JBP1, and CARM1, as well as the Saccharomyces Rmt1, have histone methyltransferase activity (McBride and Silver, 2001). However, the catalytic mechanism for the methyl group transfer as well as the makeup of the active sites of PRMTs differ somewhat from SET domain proteins.
A. Structure The catalytic domain of the PRMTs is highly conserved and consists of about 310 amino acids constituting a SAM binding domain and substrate
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binding domain (Branscombe et al., 2001; Weiss et al., 2000; Zhang et al., 2000). In addition to this conserved region, each PRMT has a unique region N-terminal to the core. Although not much is known about the function of these unique domains, it is believed that they are involved in substrate specificity and targeting. The N-terminal region of the core contains a mixed / SAM binding and methyltransferase domain. The mixed / SAM binding domain, known as the Rossman fold (Rossmann et al., 1974), is conserved among many known methyltransferase structures. Within this domain, SAM makes extensive contacts with several highly conserved residues in the I and post-I motifs (Fig. 7). The methionine group of AdoMet interacts with a conserved arginine and aspartate within the catalytic core (Zhang et al., 2000). Also, the ribose ring makes Van der Waals interactions with a glycine found in the loop between the first beta strand and the second
Fig. 7. Sequence alignment of known protein arginine methyltransferases (PRMTs). Sequences essential for AdoMet and substrate binding are indicated by blue and red bars.
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helix of the core (Zhang et al., 2000). One of the most critical interactions for PRMT activity concerns the orientation of AdoMet in the binding pocket. The AdoMet molecule is secured in this pocket by a hydrophobic interaction between the adenine ring and the phenyl ring of a phenylalanine from the N terminus of the core (Zhang et al., 2000). Aside from the conserved N-terminal region of the core found in many methyltransferases, PRMTs contain two additional -helices that precede the mixed / structure (Zhang et al., 2000). One of these helices contains the phenylalanine residue that secures AdoMet in the binding site, indicating that these extra helices may allow access for AdoMet and AdoHcy binding and exchange (Zhang et al., 2000). Also contained in the AdoMet binding region is a 12-residue loop, termed the double-E loop, which contains two completely conserved glutamates that are part of the active site (Zhang et al., 2000). The C terminus of the core contains a -barrel believed to be involved in substrate binding. Within this barrel is a highly conserved loop (named the THW loop for the conserved residues at its center), which is in very close proximity to the double-E loop of the N-terminal core (Zhang et al., 2000). These two loops are the most conserved regions of the enzymes and constitute the active site. The loops are further stabilized by a region of -sheet that makes several contacts with the loops, forming a ‘‘floor’’ for the active site (Zhang et al., 2000). These contacts and the residues involved are absolutely conserved between known histone PRMTs (Fig. 7), and are essential for the formation of the active site. Also contained in the -barrel structure of many PRMTs is a helix-turnhelix ‘‘antenna’’ motif that is involved in the formation of PRMT homodimers or homo-oligomers (Weiss et al., 2000). This dimerization usually occurs via hydrophobic contacts between the helix-turn-helix motif of one monomer and the N-terminal SAM binding domain of another (Weiss et al., 2000; Zhang et al., 2000). Several PRMTs have been shown to form either dimers or oligomers through this interaction, a process that is essential to their methyltransferase activity (Teyssier et al., 2002).
B. Catalytic Mechanism The conserved glutamates that give rise to the double-E loop interact with the guanido nitrogens of the target arginine residue. This interaction serves to redistribute the positive charge of the -nitrogens, leading to the deprotonation of one group and to priming it as a nucleophile (McBride and Silver, 2001). The deprotonated nitrogen makes a nucleophilic attack on the nearby methyl group of AdoMet. As mentioned earlier, differences between the type I and type II PRMTs determine the next methylation
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step, either catalyzing the dimethylation of one nitrogen or allowing the symmetric methylation of both guanido groups (Branscombe et al., 2001). In both types of enzymes, however, the proton stripped from the nitrogen is dispersed through a histidine–aspartate proton relay system and released into the surrounding matrix (Fersht and Sperling, 1973). The residues involved in this mechanism are completely conserved in PRMTs and are essential to the function of the enzymes. This importance is underscored by the finding that mutation of either glutamate in the double-E loop results in an inactive enzyme.
C. Identified Arginine Target Residues The known histone arginine methylation targets known so far are R17 and R26 of histone H3, and R3 of H4. JBP1/PRMT5 has been shown to methylate histone H2A and H4, although the sites of these modifications remain unknown (Pollack et al., 1999). CARM1 (cofactor associated arginine methyltransferase 1) is the enzyme responsible for the methylation of R17 and R26 of histone H3. Experiments have shown that CARM1 is recruited to the promoter regions of specific genes, and the ensuing methylation of histone arginine residues in these regions coincides with transcriptional activation (Chen et al., 1999; Schurter et al., 2001). Thus, CARM1 is recognized as a transcriptional coactivator. Histone H4 is the substrate for the type I enzymes Rmt1 (yeast) and PRMT1 (mammals). Both PRMTs methylate R3 in the histone tail. In mammals, this modification also results in transcriptional activation, as R3 methylation of histone H4 results in subsequent acetylation of the H4 tail (Wang et al., 2001). It should also be noted that both CARM1 and PRMT1 have other substrates besides histone proteins, and both of these enzymes have been shown to function as coactivators of nuclear hormone receptors, further reinforcing their role in transcriptional activation. As mentioned before, Janus kinase-binding protein 1 (JBP1/PRMT5) has been shown to methylate histones H2A and H4. Unfortunately, the sites of these modifications are as of yet unknown. However, it is known that JBP1 is the only PRMT yet capable of performing symmetric dimethylation of substrate arginine residues (Branscombe et al., 2001). It will be interesting to see the functional significance of this modification.
IV. Epilogue Histone modifications by methylation have been demonstrated to play a pivotal role in the regulation of chromatin dynamics and gene expression. Several proteins to date have been identified as functioning as histone
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methyltransferases with different substrate specificity. The wealth of other uncharacterized Set-domain containing proteins in the database has set the stage for characterization of the role of such enzymes. What remains unclear? Are histone proteins the only substrate for these Setdomain-containing proteins? Furthermore, we do not know how monomethylation, dimethylation, and trimethylation function of such methyl transferases are molecularly regulated. Most important, what are the biological consequences of histone or protein methylation in developmental regulation or even in pathogenesis of human diseases? What is clear is that the next several years will also bring a watershed of information regarding the molecular regulation and the consequences of protein modification by methylation.
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*Department of Biochemistry, Saint Louis University School of Medicine, À St. Louis, Missouri; The Saint Louis University Cancer Center, Saint Louis University School of Medicine, St. Louis, Missouri
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . II. Lysine Methyltransferases . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . A. SET Domain Containing Lysine-Specific Histone Methyltransferases . .. . . . . . B. Non-SET Domain Containing Lysine Methyltransferases. . . . . . . . . . . . . . . . .. . . . . . III. Arginine Methyltransferases . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . A. Structure . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . B. Catalytic Mechanism. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . C. Identified Arginine Target Residues . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . IV. Epilogue . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .
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I. Introduction During development, cells become committed to different fates, in part through heritable, quasi-stable changes in gene expression. The mechanisms by which the 2-m-long DNA of eukaryotic organisms is packaged into the cell nucleus while remaining functional still remains not well understood. However, the last few years have been a watershed for understanding the first stage in this packaging process: the formation of the nucleosome core particle. Nucleosomes were first observed by viewing electron micrographs of lysed nuclei (Kornberg, 1974). The chromatin appears as a series of ‘‘beads on a string,’’ the beads being the individual nucleosomes and the ‘‘string’’ the linker DNA. Since the discovery of the ‘‘beads on a string,’’ it has been demonstrated that each nucleosome consists of eight core histone proteins (two each of H3, H4, H2A, and H2B; Luger et al., 1997). Each of the core histones contains a shared region of homology termed the histone fold, and it is this motif that allows for histone–DNA contacts and for dimerization of histones (Arents and Moudrianakis, 1995). Two H3/H4 heterodimers interact via a four-helix bundle formed between the two H3 histone folds, and this tetramer is then
1 Correspondence to: Ali Shilatifard, Saint Louis University School of Medicine, Department of Biochemistry, 1402 South Grand Blvd. St. Louis, MO 63104 (e-mail: [email protected]).
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flanked by two H2B/H2A heterodimers with two four-helix bundles linking histones H2B and H4, thus bringing the core octamer together (Luger et al., 1997). The core histones are wrapped by 147 base pairs of DNA (1.65 turns around the histone octamer) in a left-handed superhelix, forming the intact nucleosome. The nucleosome structure is stabilized by about 142 hydrogen bonds between the DNA and the core histones, and several hydrophobic interactions are present between DNA and histones within the core (Luger et al., 1997). The core histones are rich in lysine and arginine residues, whose positive charge can form stable interactions with the negatively charged phosphodiester backbone of DNA. Taken together, these interactions allow for a highly stable complex to form between DNA and the core histone proteins. The nucleosome structure, when further stabilized by the linker histone H1, can compact the linear DNA about 30–40-fold, which is a significant reduction in length (Luger et al., 1997). This compaction affects the binding of nonhistone proteins, such as transcription factors, by restricting access to the binding sites within the DNA. Reduction in DNA length produced by histone-induced supercoiling in a nucleosome is significant and is an essential first step in the formation of higher-order chromatin structures. During the last decade, there has been an explosion of information regarding the role of nucleosomes in the regulation of gene expression (Workman and Kingston, 1998). Just as exciting has also been the growing possibility that the nucleosomes can transmit epigenetic information from one cell generation to the next (Jenuwein and Allis, 2001; Turner, 2002). The histone amino termini, or tails, are the sites of many of the covalent modifications that alter the nucleosome structure (Fig. 1). These tails extend away from the core of the nucleosome and are available for interactions with the DNA or with other proteins (Luger et al., 1997). There are multiple modification sites on each histone tail, and some amino acids in the histone tail can be modified in two or more ways (Schreiber and Bernstein, 2002; Turner, 2002; Figs. 1, 2). Because the sequence of the N-terminal tails is rich in arginine and lysine residues, the histone tails are very basic; covalent modification of these histone residues such as acetylation can alter the charge and structural properties of the nucleosome and can serve to restrict or allow access to the nucleosomal DNA. Recently, it has been demonstrated that modifications can also occur within the globular domain of histone H3. Gottschling and coworkers and Struhl and colleagues independently demonstrated that methylation on lysine 79 of histone H3, which occurs within the globular domain, restricts the binding of silencing proteins such as Sir2 to specific regions of the genome (Ng et al., 2002a; Van Leeuwen et al., 2002). The deletion of Dot1, which
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Fig. 1. Nucleosome structure and histone tail modification sites.
result in the loss of histone H3 methylation on lysine 79, as will be discussed later, abolishes the interaction of the silencing proteins in the body of genes and causes silencing defects at the telomers (Ng et al., 2002a; Van Leewen et al., 2002). Several known covalent modifications have been observed so far that can modify the amino acid residues in the histone tails. There include acetylation, phosphorylation, ubiquitination, and methylation. Although some of these modifications have been known for many years, only recently have functional roles for these modifications begun to surface (Workman and Kingston, 1998). Each histone can undergo numerous modifications, and the combinatorial effect of these serves to elicit a multitude of different responses. This combinatorial modification of histone tails has been referred to as the ‘‘histone code’’ (Jenuwein and Allis, 2001; Strahl and Allis, 2000) and has been proposed to play a pivotal role in the regulation of gene expression (Fig. 3). Histone acetylation was one of the first posttranslational histone proteins to be described and demonstrated to function in the regulation of transcriptional activation (Braunstein et al., 1993). To date, this modification of histone is the one best characterized (Workman and Kingston, 1998). Acetylation of a lysine residue on a histone tail can neutralize the positive charge, thus weakening the interaction of the nucleosome with the DNA backbone. This then allows for the ‘‘remodeling’’ of the nucleosome so that the transcriptional machinery, as well as other proteins, can gain access to previously restricted sites within the DNA
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Fig. 2. Histone residues modified by methylation and the enzymes responsible for each.
(Kristjuhan et al., 2002). The acetylation of histone tail has come to be known as a hallmark of gene-specific transcriptional activation, and conversely, histone deacetylation, catalyzed by HDACs, play a pivotal role in transcriptional repression (Strahl and Allis, 2000; Utley et al., 1998; Wolffe, 2001; Zhou et al., 2002). HDACs are also found in large protein complexes that function specifically at promoters. Phosphorylation of histone tails has recently been discovered as another covalent modification of histones. The covalent modification of histones by phosphorylation has been demonstrated to be involved in DNA repair, chromosomal condensation, apoptotic signaling, and heat shock–induced pathways (Ballal et al., 1975; Berger, 2001; Hunt and Dayhoff, 1977; Nowak and Corces, 2000; Rogakou et al., 2000; Van Hooser et al., 1998). Interestingly, phosphorylation of serine 10 of histone H3, catalyzed by the protein kinases Ipl1, Rsk2, and Msk1 (Berger, 2001), also alter chromatin structure and function and affect transcriptional activation, condensation of chromosomes during mitosis and meiosis, and regulation of cell division.
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Fig. 3. General information for known SET domain and PRMT proteins (* undefined).
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Histone ubiquitination was discovered as a modification of histone H2A more than 20 years ago (Ballal et al., 1975; Hunt and Dayhoff, 1977). Only recently, though, has a functional role for the ubiquitination of histone proteins been described. In Saccharomyces cerevisiae, it has been demonstrated that Rad6 is involved in the attachment of ubiquitin to lysine 123 of histone H2B (Robzyk et al., 2000). This modification of histone H2B is required as a signal for histone methylation on lysines 4 and 79 of histone H3 (Dover et al., 2002; Ng et al., 2002a, 2002b; Sun and Allis, 2002). Rad6 has been demonstrated to be involved in several different pathways such as DNA repair (Jentsch et al., 1987), proteolytic degradation (Dohmen et al., 1991), and transcriptional silencing (Huang et al., 1997). Therefore, Rad6 requires an specific E3 ligase to direct it only to its role in transcription (Wood et al., 2003). Several lines of evidence have demonstrated that Bre1 is the E3 ligase for Rad6 in transcription. First, Bre1 is found in a macromolecular complex with Rad6; second, Bre1 is required for the ubiquitination of histone H2B in vivo, which seems to be a signal for methylation of histone H3 at its 4th and 79th lysines; third, Bre1 is required for telomeric silencing; fourth, Bre1 is recruited with Rad6 to a promoter; fifth, Bre1 is essential for the recruitment of Rad6 to chromatin at a promoter; and sixth, Bre1 is dedicated to the transcriptional regulatory role of Rad6. Much like histone ubiquitination, histone methylation was discovered several decades ago, but the biological significance of this modification remained elusive. However, its functional role in the regulation of gene expression is now becoming clearer. The attachment of methyl groups to histone proteins occurs predominantly at specific lysine or arginine residues on histones H3 and H4 (Jenuwein and Allis, 2001; Schreiber and Bernstein, 2002; Stallcup, 2001; Turner, 2002; Figs. 1, 2). This modification, unlike acetylation, phosphorylation, and ubiquitination, is stable. Although deacetylases, phosphatases, and deubiquitinating enzymes have all been described, as of yet there is no known demethylase that can function on methylated histone. We have recently demonstrated that an RNA polymerase II elongation factor, the Paf1 complex, is required for the recruitment of histone methyltransferases to the elongating RNA polymerase, and have therefore suggested that it is possible that modification by methylation may serve as a mark of transcriptional memory for transcribed genes. To date, two types of histone methylation have been observed: methylation of the "-amino group of lysine, and methylation of the N nitrogens of the arginine side chain guanido group (Aletta et al., 1998; Rea et al., 2000).
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II. Lysine Methyltransferases A. SET Domain Containing Lysine-Specific Histone Methyltransferases The first histone methyltransferase to be identified was the product of the Drosophila suppressor of position-effect variegation gene, Su(var)3-9, Clr4+ (cryptic locus regulator) in Schizosaccharomyces pombe, and SUV39H1 and SUV39H2 in human (Rea et al., 2000). Su(var)3-9 contains a SET domain, a highly conserved motif originally identified at the C-terminal ends of Su(var)3-9, enhancer of zeste (a member of the Polycomb family), and Trithorax, three gene-regulatory factors in Drosophila (Dorn et al., 1993; Jones and Gelbart, 1993; Tschiersch et al., 1994; reviewed in Alvarez-Venegas and Avramova, 2002). In yeast, the product of the SET1 gene is found in a macromolecular complex called COMPASS (complex of proteins associated with Set1; Miller et al., 2001; Nagy et al., 2002; Roguev et al., 2001). COMPASS can methylate the fourth lysine of histone H3 and thereby effect regulation of transcription of genes located near chromosome telomeres. To date, COMPASS is the only histone methyl transferase that was biochemically isolated and that functions in a macromolecular complex (Krogan et al., 2002). The yeast Set1 protein alone is not capable of methylating histone H3 and requires the presence of other components of COMPASS for its enzymatic function. It has been proposed that components of COMPASS may function either in properly positioning the catalytic domain of Set1 or in proper substrate recognition by Set1 (Krogan et al., 2002). On the basis of homology of the conserved SET domains, this family of proteins is classified into four families: Su(var)3-9, E(z), TRX, and ASH1 (Alvarez-Venegas and Avramova, 2002; Jenuwein et al., 1998). Since its initial discovery, the SET domain has been described as part of several proteins involved in modulating gene activity in eukaryotes ranging from yeast to humans (Alvarez-Venegas and Avramova, 2002; Jenuwein et al., 1998; Nislow et al., 1997). This modulation happens via the histone methyltransferase activity found in many SET domain proteins and the complexes containing them. These SET domain-containing protein complexes have a number of identified lysine residue targets, and as we will describe later, the methylation of each different residue can elicit an array of different cellular responses. Because of their fairly recent discovery, only a few SET domain-containing enzymes and their target residues have been identified and crystallized (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). To date, all of the characterized SET containing proteins have been
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demonstrated to have a highly conserved active site and a binding site for S-adenosyl-L-methionine (SAM domain).
1. Structure The histone methltransferase activity (HMTase) of the SET-domaincontaining protein resides within the SET domain. The SET domain itself is approximately 130 amino acids in length (Fig. 4; Jenuwein et al., 1998). Although some areas of the sequence are highly conserved between species, others are much more varied. The pre-SET and post-SET domains can differ in sequence and structure between different HMTases. However, members of the same SET domain protein families have high
Fig. 4. Sequence alignment of some known lysine methyltransferases. The residues involved in the catalytic domain ‘‘knot’’ are indicated by red bars. The conserved active site tyrosine residue is denoted by *.
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homology in these pre-SET and post-SET domains. In each case, however, the pre-SET and post-SET domains are required for methyltransferase activity (Jacobs et al., 2002; Min et al., 2002; Rea et al., 2000; Wilson et al., 2002; Zhang et al., 2002). In many cases, these pre-SET and post-SET domains intertwine with the SET domain itself to alter the tertiary structure of the enzymes (Min et al., 2002). These variations lend different site specificities to each enzyme for methyl group attachment. However, the catalytic core of the enzyme and the SAM substrate binding site remain highly conserved. In the Su(var)3-9 and E(z) families of HMTases, the pre-SET region is rich in cysteine residues that form a triangular zinc cluster that acts to bind zinc ions very tightly, an interaction that serves to stabilize the structure (Min et al., 2002). This stabilization and positioning of the zinc cluster is essential for activity, as mutations in conserved residues that serve to position the cluster result in an inactive enzyme (Min et al., 2002). Enzymes containing this zinc cluster are also sensitive to metal chelating agents such as EDTA (Min et al., 2002; Zhang et al., 2002). Although some TRX family members also contain cysteine-rich regions N-terminal to the SET domain, these regions are at a considerable distance from the SET domain itself and may serve other functions. The members of the Su(var)3-9 family of HMTs are the only SET-domain-containing enzymes shown to have a cysteine-rich post-SET domain. Apart from the zinc clusters, many of the identified HMTases have similarities in the region N-terminal to the SET domain. The crystal structure of SET7/9 reveals the presence of an acidic groove on the pre-SET domain surface of the protein, which extends to the SET domain itself (Wilson et al., 2002). This groove may serve as the binding site for the basic N-terminal tails of histone proteins, and mutations of residues within the groove result in aberrant substrate binding. The place in which pre-SET groove intersects the SET domain also happens to be where the adenine moiety of AdoMet binds (Wilson et al., 2002). However, it should be noted that several other grooves on the surface of the SET domain surface approach the AdoMet binding site and that, as of yet, it is unclear which is responsible for histone tail binding. Although the adenine ring of AdoMet makes several contacts with the enzyme, it appears that the sulfonium component does not interact with the binding site (Wilson et al., 2002). However, the ribose ring forms Van der Waals interactions with a conserved residue in the core of the SET domain, as will be discussed later. The rest of the AdoMet binding site is a region of high sequence conservation, and the contacts between the site residues and AdoMet rely solely on hydrogen bonding. In fact, the amine nitrogen of AdoMet is hydrogen bonded to a highly conserved histidine residue on the ‘‘loop’’ of the active site knot (discussed later), and mutation of this residue abolishes enzyme activity (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002).
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The catalytic core of the SET domain is rich in -strands that comprise several regions of -sheets. The actual number of -strands may vary between different enzymes (eight and nine in Clr4 and SET7/9, respectively), but the number of alpha helices remains constant at two. In many cases, -strands found in the pre-SET region will form -sheets with the -strands of the SET domain, imparting slight variations to the SET domain structure. These small changes alter the target residue site specificity for methylation and allow the SET domain methyltransferases to target many different residues. This interplay between the pre-SET domain and the catalytic core is critical for enzyme function, and it has been demonstrated that both the pre-SET and post-SET regions that flank the SET domain are required for histone methyltransferase activity. The C-terminus region of the SET domain makes an unusual turn that threads back through a loop formed between a -strand and an alpha helix at the core of the domain to form a ‘‘knot’’ (Jacobs et al., 2002). This knot structure is the catalytic core of the enzyme. Conserved residues in the loop and C-terminal ‘‘thread’’ combine to establish a hydrophobic core in the knot, which serves to stabilize the structure. Further reinforcement of the active site structure is achieved by hydrogen bonding between several conserved residues within the knot (Fig. 4; Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). Both the hydrophobic core of the active site and the residues involved in the hydrogen bonding constitute regions of high sequence conservation. This ‘‘knotted’’ catalytic core is unique to SET domain containing enzymes and does not resemble any other previously characterized methyltransferases that use AdoMet as a methyl group donor. Within this core, AdoMet and the substrate lysine are brought into close proximity. The core of each SET domain protein contains a completely conserved tyrosine residue, which is critical to the active site function. The conserved tyrosine forms Van der Waals interactions with the ribose of AdoMet and also serves to deprotonate the "-amino group of the target lysine residue (Jacobs et al., 2002). This is critical for activity, as mutation of the tyrosine results in an inactive enzyme (Wilson et al., 2002). Note once again that the conserved residues that are critical for the catalytic ability of the SET domain are derived from the knot itself; thus, the knot structure is crucial for the formation of the active site and the activity of the SET domain itself.
2. Catalytic Mechanism of the SET Domain When AdoMet and the lysine of the substrate histone tail are bound and properly oriented in the catalytic pocket of the SET domain, the conserved tyrosine residue acts to deprotonate the "-amino group of the lysine
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Fig. 5. Catalytic data of characterized histone methyltransferases (* undefined value).
residue (Trievel et al., 2002; Fig. 6). This primes the lysine side chain to make a nucleophilic attack on the methyl group of the AdoMet molecule. The methyl group is transferred to the lysine side chain, resulting in the formation of methylated histone and the byproduct of AdoMet demethylation, AdoHcy (Jacobs et al., 2002; Min et al., 2002; Wilson et al., 2002; Zhang et al., 2002). In vitro catalytic data have been obtained for only a few of the characterized SET domain containing proteins. These data are summarized in Fig. 5. Most of the examined HKMTs have optimal methyltransferase activity between a pH of 8 and 10. The mechanism for methyl group attachment to lysine residues is illustrated in Fig. 6.
3. Identified Lysine Target Residues To date, the lysine residue methylation of histone proteins by SETdomain-containing enzymes occurs specifically at Lys4, Lys9, Lys27, and Lys36 of histone H3, and Lys20 of H4, and each lysine residue can be methylated up to three times. As more SET-domain proteins are characterized, it is possible that more histone and nonhistone targets will be found as well. For the residues that have been established as targets for KHMTs, though, a number of biological consequences for their methylation such as transcriptional silencing and activation, have been observed (Fig. 3). What is interesting about some of these methyltransferases is that they are capable of methylating the same lysine residue once, twice, or three times. Studies performed in Kouzarides’s laboratory have demonstrated that dimethylated K4 of histone H3 is associated with active euchromatic regions, but not in silent heterochromatic sites (Santos-Rosa et al., 2002). They have also demonstrated that Set1 subunit of COMPASS can catalyze dimethylation and trimethylation of K4. This correlates with the activity of many genes. They have also developed antibodies that discriminate between the di- and trimethylated state of lysine 4 of histone H3 and have demonstrated that dimethylation occurs at both inactive and active euchromatic genes, whereas trimethylation is present exclusively at active genes.
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Fig. 6. Mechanism of methyl group transfer to lysine residues.
What remains to be determined is how COMPASS dimethylates at some sites and trimethylates at other sites. It is possible that either the subunits of COMPASS or other proteins within the yeast genome can participate in this process. Lysine 9 of histone H3 is the target of many SET-domain-containing enzymes (Fig. 2). Most of the HKMTs that methylate this residue belong to the Su(var)3-9 family. In Drosophila and humans, methylation of this lysine provides a binding site for the chromo domain of heterochromatin protein 1 (HP1; Aagaard et al., 2000; Loyola et al., 2001). Work done in Drosophila has demonstrated that the interaction of HP1 and methylated
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H3 K9 is localized to regions of heterochromatin on polytene chromosomes, and alteration of HP1 recruitment affects heterochromatic gene silencing (Jacobs et al., 2001). Methylation of H3 lysine 9 then may serve to mediate heterochromatin assembly and gene silencing through its recruitment of HP1 (Hall et al., 2002; Schotta et al., 2002). The methylation of lysine 4 of histone H3 has also been extensively studied. Although the methylation of lysine 9 in higher eukaryotes is strongly related to transcriptional repression, methylation of histone H3 lysine 4 seems to antagonize this effect. In mammals, SET7/9 specifically methylates lysine 4, and this methylation coincides with transcriptional activation (Nishioka et al., 2002). SET7/9 also lacks the cysteine-rich domains found in lysine 9-specific methyltransferases, once again demonstrating the role of the pre-SET and post-SET domains in substrate specificity (Rea et al., 2000; Wilson et al., 2002). In S. cerevisiae, the methylation of K4 is carried out by COMPASS, a large SET-domain-containing protein. In contrast to the Lys4 methylation in higher eukaryotes, SET1-dependent methylation is involved with transcriptional repression at the telomeres and with mating type and rDNA loci (Bryk et al., 2002; Krogan et al., 2002; Miller et al., 2001). Recently, it was demonstrated that methylation of lysine 79 of histone H3 can regulate telomere-associated gene silencing by restricting the binding of silencing proteins to the telomeres and other ‘‘silenced’’ regions of chromatin (Van Leeuwen et al., 2002). It is also very possible that methylation of histone H3 on lysine 4 may function via a similar mechanism.
B. Non-SET Domain Containing Lysine Methyltransferases A novel histone methyltransferase was recently described as having specificity for lysine 79 of histone H3 (Ng et al., 2002a; Van Leeuwen et al., 2002). This enzyme, named Dot1 (disruptor of telomeric silencing 1) was originally discovered based on a high–copy suppressor screen of telomeric silencing (Singer et al., 1998). It was recently demonstrated that the target—a lysine residue by Dot1—is not found in the tail region of the histone but, rather, in the globular core of the histone itself. The residues are located on the top and bottom of the nucleosome core (Van Leeuwen et al., 2002). To date, Dot1 is the only enzyme known to methylate residues within the histone core. In vitro assays have revealed another interesting characteristic of Dot1. Almost all other HMTs can methylate free histone, some even being able to methylate peptides having homology to their respective target sequence. Dot1, however, will only methylate if the nucleosome is intact. Most interestingly, it was recently demonstrated that
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histone methylation by COMPASS requires histone ubiquitination by Rad6 and its E3 ligase Bre1 (Ng et al., 2002a, 2002b; Wood et al., 2003). This modification of histone H2B by ubiquitination is also a required modification for Dot1’s function: linking the molecular function of a non-SET domain and a SET domain containing methyltransferase.
1. Structure Dot1, unlike other known lysine methyltransferases, does not contain a SET domain. Rather, the methyltransferase fold resembles those of known arginine methyltransferases. In fact, many of the conserved residues found in PRMTs that are important for methylation activity are found in Dot1, and like the other known HMTs, Dot1 uses AdoMet as a methyl donor. Very recently, the crystal structure of human Dot1 in complex with AdoMet was resolved (Min et al., 2003). The N terminus of hDot1 contains the active site, and the N-terminal and C-terminal domains of the Dot1 catalytic domain are linked by a loop that also serves as part of the AdoMet binding site. The loop, and thus the SAM binding site, is highly conserved between Dot1 and other AdoMet binding proteins. Mutations within this conserved domain disrupt AdoMet binding and methyltransferase activity. The SAM binding site also contains a channel positioned near the methyl group, possibly for the binding of substrate lysine residues. Mutations within this channel do in fact abolish methyltransferase activity without affecting the overall structure of the protein (Min et al., 2003). The structure of the C terminus of Dot1 was not resolved. However, because of its distance from the active site, it is believed to be important for the substrate specificity and binding of Dot1. This region carries a positive charge, which would allow for a favorable interaction with the negatively charged backbone of DNA (Min et al., 2003). This may also explain why Dot1 is only able to methylate histone H3 in the context of an intact nucleosome.
2. Identified Lysine Target Residues The methylation of H3 K79 catalyzed by Dot1 is important for silencing at the telomeres and is believed be involved in the recruitment of Sir2 and Sir4 to these regions of DNA. A proposed mechanism is that histone methylation by Dot1 restricts Sir protein binding to the telomeres, and loss or overexpression of Dot1 function allows for promiscuous binding of the Sir proteins to chromatin within the genome, effectively titrating them away from the telomeres (Ng et al., 2002a; Van Leeuwen et al., 2002). The loss of the Sir proteins from the telomeres confers silencing defects to Dot1 deficient strains, and thus, methylation of Lysine 79 serves to restrict silencing to specific regions of the genome.
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III. Arginine Methyltransferases The methylation of arginine residues of histone proteins has also received much attention in recent years. However, not much is known about the biological significance of this process as of yet. Recent experiments have identified three distinct types of methylation that occur at arginine residues on histone tails along with two types of PRMTs that accomplish this task. To date, the three known products of arginine methylation are NG-monomethylarginine, NGNG-symmetric dimethylarginine (in which both guanido nitrogens are methylated), or NGN’G-asymmetric dimethylarginine (in which only one guanido nitrogen receives two methyl groups; McBride and Silver, 2001; Stallcup, 2001). The two types of PRMTs are classified by the type of methylation they accomplish. Type I PRMTs (PRMT1, PRMT3, CARM1/PRMT4, and Rmt1/Hmt1) produce monomethylarginine and asymmetric dimethylarginine (Chen et al., 1999; Frankel and Clarke, 2000; Gary et al., 1996; McBride et al., 2000) whereas type II methyltransferases (JBP1/PRMT5), have the ability to form monomethyl or symmetric dimethylarginine (Branscombe et al., 2001). These differences arise from variations in the arginine binding pocket, which can accommodate a previously methylated guanido group (type II) and then allow the other to be modified (symmetric), or a pocket that only allows one of the guanido nitrogens to be modified (asymmetric type 1; Branscombe et al., 2001). Like the SET domain containing methyltransferases, PRMTs require AdoMet as a cofactor and methyl group donor. The PRMTs identified so far exist as members of multisubunit complexes, the formation of which is critical for in vivo methyltransferase activity (Teyssier et al., 2002). Many of the arginine methyltransferases also form homo-dimers or homooligomers, a step that is required for their catalytic activity (Weiss et al., 2000). This need to oligomerize has not been demonstrated for any known lysine methyltransferases. The identities of several protein arginine methyltransferases are now known, but only a few have been shown to have specificity for histone proteins. The mammalian PRMT1, JBP1, and CARM1, as well as the Saccharomyces Rmt1, have histone methyltransferase activity (McBride and Silver, 2001). However, the catalytic mechanism for the methyl group transfer as well as the makeup of the active sites of PRMTs differ somewhat from SET domain proteins.
A. Structure The catalytic domain of the PRMTs is highly conserved and consists of about 310 amino acids constituting a SAM binding domain and substrate
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binding domain (Branscombe et al., 2001; Weiss et al., 2000; Zhang et al., 2000). In addition to this conserved region, each PRMT has a unique region N-terminal to the core. Although not much is known about the function of these unique domains, it is believed that they are involved in substrate specificity and targeting. The N-terminal region of the core contains a mixed / SAM binding and methyltransferase domain. The mixed / SAM binding domain, known as the Rossman fold (Rossmann et al., 1974), is conserved among many known methyltransferase structures. Within this domain, SAM makes extensive contacts with several highly conserved residues in the I and post-I motifs (Fig. 7). The methionine group of AdoMet interacts with a conserved arginine and aspartate within the catalytic core (Zhang et al., 2000). Also, the ribose ring makes Van der Waals interactions with a glycine found in the loop between the first beta strand and the second
Fig. 7. Sequence alignment of known protein arginine methyltransferases (PRMTs). Sequences essential for AdoMet and substrate binding are indicated by blue and red bars.
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helix of the core (Zhang et al., 2000). One of the most critical interactions for PRMT activity concerns the orientation of AdoMet in the binding pocket. The AdoMet molecule is secured in this pocket by a hydrophobic interaction between the adenine ring and the phenyl ring of a phenylalanine from the N terminus of the core (Zhang et al., 2000). Aside from the conserved N-terminal region of the core found in many methyltransferases, PRMTs contain two additional -helices that precede the mixed / structure (Zhang et al., 2000). One of these helices contains the phenylalanine residue that secures AdoMet in the binding site, indicating that these extra helices may allow access for AdoMet and AdoHcy binding and exchange (Zhang et al., 2000). Also contained in the AdoMet binding region is a 12-residue loop, termed the double-E loop, which contains two completely conserved glutamates that are part of the active site (Zhang et al., 2000). The C terminus of the core contains a -barrel believed to be involved in substrate binding. Within this barrel is a highly conserved loop (named the THW loop for the conserved residues at its center), which is in very close proximity to the double-E loop of the N-terminal core (Zhang et al., 2000). These two loops are the most conserved regions of the enzymes and constitute the active site. The loops are further stabilized by a region of -sheet that makes several contacts with the loops, forming a ‘‘floor’’ for the active site (Zhang et al., 2000). These contacts and the residues involved are absolutely conserved between known histone PRMTs (Fig. 7), and are essential for the formation of the active site. Also contained in the -barrel structure of many PRMTs is a helix-turnhelix ‘‘antenna’’ motif that is involved in the formation of PRMT homodimers or homo-oligomers (Weiss et al., 2000). This dimerization usually occurs via hydrophobic contacts between the helix-turn-helix motif of one monomer and the N-terminal SAM binding domain of another (Weiss et al., 2000; Zhang et al., 2000). Several PRMTs have been shown to form either dimers or oligomers through this interaction, a process that is essential to their methyltransferase activity (Teyssier et al., 2002).
B. Catalytic Mechanism The conserved glutamates that give rise to the double-E loop interact with the guanido nitrogens of the target arginine residue. This interaction serves to redistribute the positive charge of the -nitrogens, leading to the deprotonation of one group and to priming it as a nucleophile (McBride and Silver, 2001). The deprotonated nitrogen makes a nucleophilic attack on the nearby methyl group of AdoMet. As mentioned earlier, differences between the type I and type II PRMTs determine the next methylation
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step, either catalyzing the dimethylation of one nitrogen or allowing the symmetric methylation of both guanido groups (Branscombe et al., 2001). In both types of enzymes, however, the proton stripped from the nitrogen is dispersed through a histidine–aspartate proton relay system and released into the surrounding matrix (Fersht and Sperling, 1973). The residues involved in this mechanism are completely conserved in PRMTs and are essential to the function of the enzymes. This importance is underscored by the finding that mutation of either glutamate in the double-E loop results in an inactive enzyme.
C. Identified Arginine Target Residues The known histone arginine methylation targets known so far are R17 and R26 of histone H3, and R3 of H4. JBP1/PRMT5 has been shown to methylate histone H2A and H4, although the sites of these modifications remain unknown (Pollack et al., 1999). CARM1 (cofactor associated arginine methyltransferase 1) is the enzyme responsible for the methylation of R17 and R26 of histone H3. Experiments have shown that CARM1 is recruited to the promoter regions of specific genes, and the ensuing methylation of histone arginine residues in these regions coincides with transcriptional activation (Chen et al., 1999; Schurter et al., 2001). Thus, CARM1 is recognized as a transcriptional coactivator. Histone H4 is the substrate for the type I enzymes Rmt1 (yeast) and PRMT1 (mammals). Both PRMTs methylate R3 in the histone tail. In mammals, this modification also results in transcriptional activation, as R3 methylation of histone H4 results in subsequent acetylation of the H4 tail (Wang et al., 2001). It should also be noted that both CARM1 and PRMT1 have other substrates besides histone proteins, and both of these enzymes have been shown to function as coactivators of nuclear hormone receptors, further reinforcing their role in transcriptional activation. As mentioned before, Janus kinase-binding protein 1 (JBP1/PRMT5) has been shown to methylate histones H2A and H4. Unfortunately, the sites of these modifications are as of yet unknown. However, it is known that JBP1 is the only PRMT yet capable of performing symmetric dimethylation of substrate arginine residues (Branscombe et al., 2001). It will be interesting to see the functional significance of this modification.
IV. Epilogue Histone modifications by methylation have been demonstrated to play a pivotal role in the regulation of chromatin dynamics and gene expression. Several proteins to date have been identified as functioning as histone
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methyltransferases with different substrate specificity. The wealth of other uncharacterized Set-domain containing proteins in the database has set the stage for characterization of the role of such enzymes. What remains unclear? Are histone proteins the only substrate for these Setdomain-containing proteins? Furthermore, we do not know how monomethylation, dimethylation, and trimethylation function of such methyl transferases are molecularly regulated. Most important, what are the biological consequences of histone or protein methylation in developmental regulation or even in pathogenesis of human diseases? What is clear is that the next several years will also bring a watershed of information regarding the molecular regulation and the consequences of protein modification by methylation.
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