Histone methylation versus histone acetylation: new insights into epigenetic regulation

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Histone methylation versus histone acetylation: new insights into epigenetic regulation Judd C Rice and C David Allis Post-translational addition of methyl groups to the aminoterminal tails of histone proteins was discovered more than three decades ago. Only now, however, is the biological significance of lysine and arginine methylation of histone tails being elucidated. Recent findings indicate that methylation of certain core histones is catalyzed by a family of conserved proteins known as the histone methyltransferases (HMTs). New evidence suggests that site-specific methylation, catalyzed by HMTs, is associated with various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly. Taken together, these new findings suggest that histone methylation may provide a stable genomic imprint that may serve to regulate gene expression as well as other epigenetic phenomena. Addresses Department of Biochemistry and Molecular Genetics, University of Virginia, Health Sciences Center, Box 800733 Jordan Hall, Room 6222, Charlottesville, Virginia 22908-0733, USA Correspondence: C David Allis; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:263–273 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations CAF-1 chromatin assembly factor 1 CD chromodomain ChIP chromatin immunoprecipitation chromo chromatin organization modifier CSD chromo shadow domain HATs histone acetyltransferases HDACs histone deacetylases HDM histone demethylase HMTs histone methyltransferases PEV position effect variegation PMEFs primary mouse embryonic fibroblasts SET Su(var), E(z) trithorax Su(var) suppressor of variegation TXR transcriptional regulator proteins

Introduction Nucleosomes, the fundamental structural units of chromatin, are comprised of the core histone octamer (H2A, H2B, H3 and H4) and the associated DNA that wraps around them. Although the crystal structure of a nucleosome core particle has provided considerable insight into the protein–protein and protein–DNA interactions that govern nucleosome structure [1], little is known about how distinct functional domains of chromatin are established and maintained [2]. The precise organization of chromatin is critical for many cellular processes, including transcription, replication, repair, recombination and chromosome segregation. Dynamic changes in chromatin structure are directly influenced by post-translational modifications of the amino-terminal tails of the histones [3,4]. These highly

basic histone tails are predicted to be less structured than the histone fold regions and are believed to interact with the negatively charged DNA backbone or with other chromatin-associated proteins [2,3,5–9]. Specific amino acids within these histone tails are targets for a number of post-translational modifications, including acetylation, phosphorylation, poly(ADP-ribosylation), ubiquitination and methylation [10–12]. These covalent modifications may alter the interaction of the histone tail with DNA or with chromatin-associated proteins that may be required for different downstream cellular processes [13•,14•,15]. Histone methylation was first discovered more than thirtyfive years ago [16]. Until quite recently, however, little was known about the biological significance of this covalent modification due, in part, to a lack of convenient electrophoretic assays or immunological tools to detect its presence. Therefore, little information was available as to the enzyme systems responsible for the establishment and maintenance of histone methylation. Unlike the dynamic ‘on–off’ nature of histone acetylation, early studies found that mammalian histones H3 and H4 were highly methylated with little turnover of the methyl groups [17–20]. The apparent stability of this modification in bulk histone preparations led to the belief that histone methylation was a generic and static modification. In contrast, other findings hinted that histone methylation, like histone acetylation, is a dynamic process involved in a number of diverse biological processes including transcriptional regulation, chromatin condensation, mitosis and heterochromatin assembly [21–26,27••,28•,29••]. In this review we compare and contrast the histone methyllysine modification with the well studied acetyllysine modification to provide contemporary insights into the functional and biological consequences of these histone modifications. We propose that methylation of specific lysine residues in histone tails functions as a stable epigenetic mark that directs particular biological functions, ranging from transcriptional regulation to heterochromatin assembly. Furthermore, we speculate that, in certain circumstances, context-dependent histone methylation can provide a critical ‘mark’ on the histone tails, leading to the recruitment and binding of chromatin-associated proteins, which ultimately results in a distinct biological response. It should be noted that arginine residues in histone tails are also targets for in vitro methylation catalyzed by a new class of coactivators functionally linked to a variety of nuclear receptors (i.e. CARM1 and PRMT1) [27••,30,31]. This review focuses predominantly on lysine methylation, but a current review on arginine methylation of histones is highly recommended [32•].

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Histone methylation versus histone acetylation. (a) Molecular structure of lysine, acetyllysine and methyllysine. Conserved lysine residues in the amino-terminal histone tails can be posttranslationally modified by acetylation or methylation. Acetylation is catalyzed by HATs and is removed by opposing HDACs. Acetyl substitution of the ε-amino group of lysine occurs once and results in a more acidic, hydrophilic residue. Methyl substitution is catalyzed by the HMTs and may be removed by yet unidentified histone demethylases (HDMs?). It remains unclear how increasing degrees of methyl addition (mono, di and tri) is brought about and whether

the same HMT can catalyze all of these methyl additions. Nevertheless, increasing methyl addition increases the basicity and hydrophobicity of the lysine without altering its overall charge. (b) Known acetyl and methyl modifications of lysine residues in the amino-terminal tails of human histone H3 and H4 are shown. Conserved lysines are in bold and their amino-acid position in the histone tail is indicated by the numbers. The in vivo sites for lysine acetylation and methylation are shown. Current findings suggest that each lysine is either acetylated or methylated with the exception of lysine 9 in the H3 tail (see text for details).

Lysine methylation and acetylation of histones

decreases their overall positive charge (Figure 1a). As these tails are highly basic, it has long been postulated that acetylation decreases their affinity for the negatively charged DNA and facilitates the binding of proteins that regulate transcription to chromatin templates [37–40]. In addition, recent evidence suggests that histone acetylation may alter the structure of histone tails by increasing their alpha-helical content [41]. In contrast, methylation does not alter the overall charge of the histone tails, however, increasing methyl addition (mono, di or tri) does increase its basicity and hydrophobicity (Figure 1a). Furthermore, increased methyl addition on histone tails increases their affinity for anionic molecules (i.e. DNA) and results in increased resistance to trypsin digestion [42,43]. Taken together, these observations suggest a tight association between the methylated histone tail and DNA and/or chromatin. Therefore, similar to acetylation, methylation of histones may alter the interaction of the histone tails with the DNA and/or chromatin-associated proteins and, hence, nucleosomal structure and function.

The packaging of eukaryotic DNA into nucleosomal arrays presents a major obstacle to transcription that must be dealt with to allow the transcriptional machinery to access the DNA template. The discovery of enzyme complexes dedicated to chromatin remodeling, whether by directly modifying histone proteins or by ATP-dependent nucleosomal remodeling complexes, has led to new insights into the mechanisms of transcription [2,12,33,34]. Compelling evidence obtained over the past six years indicates that acetylation of core histones, which is catalyzed by the histone acetyltransferases (HATs) and removed by the histone deacetylases (HDACs) (Figure 1a), is causally linked to transcriptional activation. Interestingly, a surprisingly large number of previously identified components of the transcriptional apparatus are HATs [35,36]. Enzymatic acetylation of the ε-amino group of conserved lysine residues in the amino-terminal tails of histones

Histone methylation versus histone acetylation Rice and Allis

Methylation of histones is catalyzed by histone methyltransferases (HMTs), which use S-adenosylmethionine (SAM) as a cofactor in much the same way that HATs utilize acetyl-coenzyme A as a cofactor (Figure 1a). In contrast to acetylation, the majority of data available suggests that the methyl modification is relatively irreversible [19,43,44]. Because there is little evidence for large scale decreases in methylated histones from bulk chromatin, the existence of a global histone demethylase (HDM) seems unlikely. However, like acetylation, it is plausible that regulated or targeted demethylation of histones occurs on specific residues at specific loci or promoters, although these activities have yet to be discovered (Figure 1a).

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during DNA replication and reassembled by G2/M, concomitant with the observed peak activity of histone methylation [50]. Once established (following replication), telomeric silent chromatin is relatively stable. These early observations suggested that histone methylation, in certain circumstances, may serve as a stable epigenetic mark that aides in the establishment of discrete chromosomal regions involved in specific chromatin-mediated events. Moreover, histone methylation may have the capacity to impart different biological functions depending upon the specific methylated histone, lysine residue and chromatin-associated protein involved (see below).

Histone methylation as an epigenetic mark The major targets for post-translational acetylation and methylation are conserved lysine residues located in the amino-terminal tails of histones H3 and H4, although these modifications also occur on the H2A and H2B tails [45]. Acetylation of specific lysine residues by specific HATs is well documented [35,36]. Similarly, histone methylation is also a non-random event in vivo, as specific lysines are selectively methylated (Figure 1b; [45]). Therefore, similar to HATs, each HMT is likely to have its own unique set of kinetic parameters, as well as histone and lysine preferences. Furthermore, various methyllysine species (mono, di or tri) have been observed for each methylated lysine residue in vivo, however, the biological significance of these differences remains undetermined (Figure 1a). As depicted in Figure 1b, specific lysine residues in both the H3 and H4 tails appear to be targeted for either acetylation or methylation. However, one lysine in the H3 tail (Lys9) can be targeted for both modifications, presumably in different biological contexts (see below). It is possible that other acetylated lysines could also be targets for methylation, as early studies using bulk histone fractions would not have been able to detect minor changes in methylation. With the advent of more sensitive methods and techniques, such as mass spectrometry and immunological reagents, we look forward to a critical re-examination of this issue. Taken together, these observations suggest that certain combinations of acetyl and methyl modifications of lysines in histone tails may have antagonistic or cooperative biological effects. For example, hyperacetylated H4 from transcriptionally active chromatin preparations is a preferential target of histone H3 methylation, suggesting that these modifications may act synergistically to promote transcription in a way that remains unclear [26]. Histone acetylation occurs throughout the cell cycle, whereas histone methylation peaks in G2 phase, subsequent to DNA replication and histone synthesis, and during heterochromatin assembly [17,18,46–48]. In addition, developing rat neurons have robust HMT activity, whereas non-replicating adult neurons exhibit marked decreases in HMT activity [49]. Furthermore, using telomere position effect variegation (TPE) as a model for epigenetic silencing, data in yeast suggest that this repressive chromatin state is disassembled

Eukaryotic genomes are often conveniently described as transcriptionally active (euchromatin) or transcriptionally silent (heterochromatin). Heterochromatin was originally defined as the fraction of the genome that remained visibly condensed during interphase. More recently, heterochromatin has been defined as genomic regions that are gene poor, contain large blocks of repetitive DNA, are inaccessible to DNA-modifying reagents and replicate late in the cell cycle [51,52]. Interestingly, when a transcriptionally active gene is displaced from its normal euchromatic position to the vicinity of heterochromatin, the gene becomes and remains inactivated [53–55]. Remarkably, epigenetic inheritance of this inactivated state is propagated during mitosis and through the germ-line during meiosis [56,57]. This epigenetic phenomenon, known as position effect variegation (PEV), provides an attractive model to understand the heritable molecular imprint that specifies the transcriptional state of a gene, as well as the factors that influence its stability. Genetic screens for suppressors or enhancers of PEV in Drosophila melanogaster [53,58] and Schizosaccharomyces pombe [59,60] have identified numerous genes whose products probably stabilize and/or propagate higher-order chromatin structure. For example, about 30–40 loci, collectively referred to as the Su(var) group (suppressor of variegation) [53], include catalytic components such as HDACs, protein phosphatases and S-adenosylmethionine (SAM) synthetases [61,62], as well as heterochromatinassociated proteins, such as heterochromatin-associated protein 1 (HP1) (Su(var)2-5) [63], which is thought to play an architectural role (see below). Consistent with the paradigm established between histone acetylation and transcription, the recent discovery that a Su(var) protein is a lysine histone methyltransferase has provided a critical link between histone methylation and heterochromatin assembly [29••]. The Drosophila Su(var)3-9 protein is localized in condensed chromatin and is a key regulator in the organization of repressive chromatin; however, its precise function was not known [64]. Rea et al. [29••] demonstrated that the human and S. pombe homologues of Su(var)3–9, SUV39H1 and Clr4, respectively, are HMTs that specifically methylate H3 Lys9 in vitro. The methylation of

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Figure 2 (a)

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H3 Lys9 was catalyzed by the conserved SET domain (Su(var)3-9, E(z), trithorax) and two flanking cysteine-rich regions in SUV39H1 and Clr4 (Figure 2b). All three of these regions are required for HMT activity in vitro since other SET domain-containing proteins that lack one or more of these regions, including human EZH2 and HRX, fail to methylate histones. Interestingly, most Suv39h-null mice were non viable. The small fraction that remained viable were growth retarded compared to control mice, suggesting a role for histone methylation in normal development. Furthermore, primary mouse embryonic fibroblasts (PMEFs) derived from the Suv39h-null mice exhibited increased division defects during mitosis and had weakly defined heterochromatic regions compared to normal PMEFs. Consistent with this, overexpression of SUV39H1 induced ectopic heterochromatin formation. Taken together, these novel findings implicate H3 Lys9 methylation in the proper assembly of heterochromatin in a conserved pathway leading to epigenetic silencing.

Histone H3 Lys9 methylation, HP1 recruitment and heterochromatin assembly Several nuclear HATs (A-type) contain an evolutionarily conserved motif known as the bromodomain (Figure 2a). Recent discoveries indicate that the bromodomain and double bromodomain of PCAF and TAFII250, respectively, bind preferentially and specifically to acetylated lysines on histone tails in vitro (Figure 2a; [65••,66••]). Therefore, as predicted by the ‘histone code’ hypothesis [13•,14•],

Function of conserved motifs within certain chromatin-modifying proteins. (a) Schematic drawing of conserved motifs within the transcriptional regulators PCAF, a GCN5 homologue, and TAFII250, a subunit of the TFIID complex. PCAF and TAFII250 contain a HAT catalytic domain that acetylates specific lysine residues on the histone tails (left; not shown for TAFII250). In addition, each protein contains an evolutionarily conserved bromodomain (bromo) and double bromodomain, respectively, that binds to the appropriately acetylated lysines on the histone tails to promote transcription (right). (b) Schematic representation of the Su(var)3–9 family of HMTs and heterochromatin-associated proteins. Human SUV39H1 and fission yeast Clr4 proteins contain a conserved catalytic SET domain flanked by two cysteine-rich regions (Cys), which are required for methyltransferase activity (left). The evolutionarily conserved chromodomain (chromo) of human HP1 and fission yeast Swi6 proteins bind to the appropriately methylated histone tail (i.e. H3 Lys9) to induce the assembly of heterochromatin (right). The exact functions of the HMT chromodomain and HP1 chromo shadow domain (shadow) are not known. Note: these drawings are not to scale.

one functional consequence of histone acetylation may be that the acetyl modification serves as a ‘mark’ on the histone tail that leads to the recruitment and binding of bromodomain-containing HATs and other coactivators to chromatin for transcription [67]. These findings strongly support the theory that alterations in nucleosomal structure, induced or perturbed by covalent histone modifications, may be important in the recruitment of chromatin-associated proteins that ultimately influence distinct cellular processes [41,68•]. Recent findings indicate that a similar conserved pathway may exist for histone methylation and heterochromatin assembly, with the methyl modification serving as the ‘mark’ on the histone tails to recruit heterochromatin-associated proteins. A potential methyl-histone binding candidate is HP1 (Figures 2b and 3a). Interestingly, HP1 and its S. pombe homologue, Swi6, are required for heterochromatin formation and colocalize with SUV39H1 and Clr4, respectively [63,69,70•]. Many heterochromatin-associated proteins, like HP1 and Su(var)3-9, share a common evolutionarily conserved domain known as the chromodomain (chromatin organization modifier) (Figure 2b; [63,71]). In addition, numerous heterochromatin-associated proteins contain a single or repeated chromodomain (CD) followed by a chromo shadow domain (CSD) [72]. These domains were thought to mediate protein–protein interactions responsible for targeting these proteins to their proper chromosomal positions by mechanisms that remained unclear until recently [57,73].

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Figure 3 Temporal model of heterochromatin assembly. (a) Key players involved in heterochromatin assembly in D. melanogaster and S. pombe. The transcriptionally silent pericentric heterochromatin in flies and the silent matingtype locus (mat) and centromeric repeats (cen) in fission yeast are depicted as red bricked structures. Several well-studied, conserved chromatin-associated proteins are shown for both organisms, these are HDACs, HMTs or heterochromatin-associated proteins (e.g. HP1). *Rpd3 was previously shown to be an enhancer of PEV [61]. (b) One proposed temporal pathway leading to the establishment of transcriptionally silent heterochromatic regions with regard to the covalent modifications in the histone H3 tail. The acetyl group on H3 Lys9, a modification often associated with transcriptionally active regions, is removed by an HDAC prior to methylation by an HMT. The CD of an HP (i.e. HP1) selectively recognizes and binds to the H3 Lys9-methyl modification resulting in the self-assembly and propagation of heterochromatin and transcriptional silencing. Other trans-acting factors and covalent histone modifications are likely to influence the temporal sequence, kinetic steps and final outcome of this proposed pathway (see text for details).

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Two independent research groups have recently shown that the chromodomain, but not the chromo shadow domain, of HP1 and Swi6 preferentially bind to methylated H3 Lys9 in vitro [74••,75••]. The chromodomain of HP1 was selective for the H3 Lys9-methyl modification, as demonstrated by the lack of binding to an H3 unmodified or H3 Lys4-methyl peptide [75••]. In addition, the H3 Lys9-methyl modification appears to be highly specific for the HP1 chromodomain, as other chromodomain-containing proteins, including SUV39H1, polycomb (M33) and Mi-2, failed to bind to an H3 Lys9-methyl peptide in these assay conditions. Interestingly, HP1 chromodomain binding affinity was significantly less than that of the full-length HP1, suggesting that native HP1 (or an HP1 dimer) is required for high-affinity binding in vivo [74••,75••]. In Suv39h1-null PMEFs, HP1 is dispersed from heterochromatic regions and ‘rescue’ of SUV39H1 by retroviral expression in these cells induced localization of HP1 to heterochromatin foci [74••]. Similarly, in S. pombe, functional Clr4 protein is required for Swi6 localization and transcriptional silencing at centromeric regions

[75••]. Therefore, analogous to the bromodomain-binding acetylated histone peptides, chromodomain-containing proteins, such as HP1 and Swi6, are predicted to bind to the H3 Lys9-methyl modification catalyzed by the Su(var)3-9 family of HMTs in order to establish silent regions of heterochromatin. In S. pombe and D. melanogaster, genes known to encode heterochromatin-associated proteins have been identified and include HDACs, HMTs and HP1-like proteins (Figure 3a). Importantly, homologues of these proteins exist in yeast, flies and mammals underscoring what is likely to be a highly conserved pathway of heterochromatin assembly. As these proteins are found together in repressive complexes, it is thought that they act cooperatively to induce a silent epigenetic state that propagates along distinct chromosomal regions. However, the precise function(s) of these components and the kinetic steps leading to the initiation and maintenance of this epigenetic pathway have remained elusive until quite recently.

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Proposed pathway of heterochromatin assembly leading to epigenetic silencing As depicted schematically in Figure 3b, an understanding of what may be one major pathway of heterochromatin assembly and heterochromatin-induced gene silencing is beginning to emerge. In this model, the status of covalent modifications on the H3 tail plays a critical role in the final outcome of heterochromatin assembly, leading to an epigenetically silent state. For example, H3 Lys9 is acetylated prior to histone deposition in some species [76], suggesting that one role of HDACs in these complexes may be to deacetylate Lys9 to ‘clear’ the ε-amino group of Lys9 so that it can be methylated by members of the Su(var)3-9 family of HMTs. Conversely, acetylation of Lys9 may serve to maintain transcriptionally competent regions of the genome by blocking methylation of Lys9 and preventing the downstream assembly of heterochromatin. We envision that once the acetyl modification is enzymatically removed by HDACs, the responsible HMT is free to methylate Lys9, resulting in the recruitment of HP1. Binding of HP1 ultimately leads to the formation of heterochromatin via a self-propagating pathway that may involve dimerization of HP1 molecules through chromoshadow–chromo-shadow domain interactions. The proposed link between HP1-mediated heterochromatin assembly and H3 Lys9 methylation depicted in Figure 3 leaves many important questions unresolved. This model relies on the recognition of chromodomains in proteins such as HP1 with appropriately methylated histone tails. Whether active demethylases exist to reverse this state, or whether the long suspected methyl ‘marks’ are only removed by histone replacement or repeated rounds of DNA replication, is not clear. It also remains unclear whether H3 Lys9 is the physiological target of the SET-domain-containing HMTs in vivo [74••,75••]. In addition, Figure 3 predicts that the H3 Lys9methyl modification and HP1 modification may be localized exclusively to heterochromatic regions. If this model is accurate, it is currently unresolved where the boundaries between H3 Lys9 methylation and euchromatin begin and end. Furthermore, if HP1 localization is functionally dependent upon H3 Lys9 methylation, it is unclear how overexpression of HP1 can ‘spread’ heterochromatin [77]. This could be explained by an HP-1–HMT interaction, which would serve to recruit additional HMTs, methylate additional histones and propogate the spreading phenomenon (see also Update). Other chromatin-associated proteins or other covalent histone modifications may be required for proper heterochromatin assembly. During chromatin assembly, H4 acetylated at Lys5 and Lys12 is deposited onto newly synthesized DNA and, once deposited, these residues are usually deacetylated [14•,76]. The association of chromatin assembly factor 1 (CAF-1), deposition-related HAT complexes and certain repressive HDAC complexes containing RbAp48/p46 could play a fundamental role in heterochromatin assembly [78]. CAF-1 is a polypeptide

complex that mediates histone deposition on newly replicated DNA. The large subunit of CAF-1, p150, binds to the chromo shadow domain of HP1 and is concentrated at regions of heterochromatin [79]. Deletion of the HP1 binding region of p150 results in derepression of a transfected reporter gene in mammalian cells [80]. These data suggest that CAF-1 has a heterochromatin-associated function that may be linked to the well known late replication property of heterochromatin. RbAp48/p46 proteins appear to facilitate the nucleosome remodeling that is necessary for the efficient acetylation/deacetylation of lysines in H4 that are covered by the natural wrapping of DNA around the histone octamer [81]. It is interesting to note that H4 Lys20, which is very close to the first alpha helix of H4, is methylated late in the cell cycle, near to the G2/M boundary [41]. Whether methylation of H4 Lys20 and/or acetylation at Lys5 and Lys12 are covalent ‘markings’ that facilitate the binding of HP1 or other chromodomain-containing, heterochromatin-associated proteins is not known but remains an intriguing possibility. In addition, other covalent histone modifications, such as H3 Ser10 phosphorylation, antagonize H3 Lys9 methylation [15,29••]. Thus, it remains a formal, and likely, possibility that other transacting factors besides those outlined in Figure 3a, and other histone modifications besides those illustrated in Figure 3b, play critical, yet undetermined, roles in the overall heterochromatin assembly pathway. As immunological reagents are developed against these proteins and as physiologically relevant covalent histone modifications are identified, we look forward to in vivo tests of these and related models using chromatin immunoprecipitation (ChIP) assays (see also Update).

Histone methylation and transcriptional regulation Although transcriptional activation is typically attributed to histone acetylation, the specific acetylation of H4 Lys12 is associated with silent chromosomal regions in various organisms [82,83]. In contrast to H3 Lys9, methylation of H3 Lys4 is specifically associated with the transcriptionally active macronuclei, but not the inactive micronuclei, in Tetrahymena [28•]. Moreover, H3 Lys4-methyl modification is conserved throughout evolution. Interestingly, acetylated isoforms of H3 and H4 are the preferential targets of histone methylation, suggesting that HMTs and HATs act synergistically to promote transcription by mechanisms that remain unclear [24–26,84]. These findings suggest a correlation between H3 Lys4 methylation, histone acetylation and transcriptional competency. Further studies are required to clarify this since methylation of arginine residues in H3 and H4 have also been implicated in transcriptional regulation [32•]. In addition, histone acetyltransferase CBP/p300 (CREB-binding protein) coimmunoprecipitates with an HMT [85] and, although unidentified, the HMT is likely to be the SET domain-containing, CBP-associated ASH1 protein [86]. Interestingly, the preferred site for methylation of

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Figure 4 Histone acetylation or methylation: a matter of choice. (a) Lysine residues in the histone tails can be acetylated by a HAT that can be rapidly removed by an antagonistic HDAC (bold arrows). Following a balanced increase in histone acetylation, the acetyl modification may recruit and bind to a conserved domain within a transcriptional regulator protein (TXR), such as the bromodomain of HATs (Figure 2A). The specific acetylated lysine(s) and the biological function of the TXR determine the final outcome leading to a transcriptionally active or silenced state. (b) Prior to lysine methylation of a histone tail by an HMT, the acetyl modification must first be ‘cleared’ by an HDAC. Once methylated, a conserved domain within a TXR may bind to the methyl modification resulting in transcriptional activation or silencing depending upon the specific methylated lysine and biological function of the TXR. It is currently not known to what extent this pathway is reversible; a pathway that would require the activity of an, as yet, unknown histone demethylase (HDM?). Depending on the (ir)reversibility of pathway (b), histone methylation may lead to a more stable epigenetic state compared to histone acetylation.

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this HMT in vitro was H3 Lys9, suggesting that H3 Lys9 methylation may play a role in both transcriptional activation and silencing. Could differences in methylated species of H3 Lys9 (i.e. mono, di or tri) explain these seemingly contradictory data (Figure 1a)? Different covalent modifications, alone or in combination, on the same or different histone tails are associated with distinct cellular process by the recruitment of chromatinassociated proteins, as predicted by the ‘histone code’ [13•,14•]. The acetylation status of certain lysine residues in histone tails appears to result from the opposing activities of HATs and HDACs (Figure 4a). The acetyl modification can be rapidly turned over in some chromatin environments [2,87], suggesting that the ‘strength’ or ‘transient’ nature of gene expression may be coupled to the degree of acetylation. This acetyl modification may serve as a labile ‘mark’ permitting the binding of conserved motifs within specialized transcriptional regulator proteins (TXR), such as the bromodomain(s) within PCAF or TAFII250 (Figure 2a). The biological consequences of this interaction would be dependent upon the specific acetylated lysine residue(s) and the inherent properties of the TXR, resulting in either transcriptional activation or silencing. For example, the double bromodomain of the TAFII250 HAT binds to diacetylated Lys5/Lys12 or Lys8/Lys16 in histone H4 resulting in transcription. In contrast, H4 Lys12 acetylation is coincident with silencing, however, the chromatin-associated protein and its conserved binding motif are currently unknown.

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For histone methylation to take place, we envision that the target lysine residue must first be cleared of any other preexisting modifications, for example, acetylation (Figure 4b). Because no HDMs have yet been identified, methylation of histones may serve as a more stable epigenetic mark, in contrast to the rapidly turned-over populations of acetylated chromatin. Once methylated by an HMT, the methyl ‘mark’, like the acetyl ‘mark’, may bind to conserved motifs within specialized TXRs, such as the chromodomain of HP1. Again, the effects of this interaction on transcription would be dependent upon the specific methylated lysine residue(s) and the inherent properties of the TXR (Figure 4b). For example, the chromodomain of HP1 binds to the H3 Lys9-methyl ‘mark’, resulting in heterochromatin assembly and epigenetic silencing. In contrast, H3 Lys4 methylation appears to be associated with transcription, however, the chromatin-associated protein and its conserved binding motif are currently unknown. Although not all chromodomain-containing proteins bind to H3 Lys4 or Lys9 methylated peptides in vitro [74••,75••], we note that there are chromodomain-containing HATs that seem to facilitate transcription, such as MOF and Esa1 [35,36]. Could the chromodomains of these proteins bind to methylated histones in vivo to promote transcription and, if so, which methylated lysines do they bind? In addition, there are other chromodomain-containing proteins associated with repressive chromatin remodeling complexes, such as Mi-2 of NuRD [88–90]. Interestingly, the Mi-2 ATPase subunit of NuRD contains

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a double chromodomain and is thought to be associated with transcriptional silencing by DNA methylation (see below). Analogous to the double bromodomain of TAFII250 (Figure 2a), we wonder to what extent the double chromodomain of Mi-2 might selectively bind to appropriated methylated histone tails. We also wonder if this double chromodomain may exhibit a preference for the appropriately dimethylated lysines within the same or distinct histone tails.

Histone methylation and potential links to DNA methylation and cancer The methylation of cytosines in CpG island promoters is associated with the transcriptional silencing of human tumor suppressor genes by the recruitment of methyl-binding proteins (MBPs) and their associated repressive complexes [91]. Some of these repressive complexes, such as Sin3A and NuRD, contain HDACs that deacetylate H3 and H4 [90,92]. Although a few transcriptionally silenced tumor suppressor genes in cancer cell lines can be partially reactivated in the presence of a DNA methylase inhibitor and histone deacetylase inhibitor, complete reactivation is rarely achieved [93]. On the basis of these observations, we speculate that these MBP-associated repressive complexes may contain HMTs that serve to ‘lock’ the promoter and chromatin into a transcriptionally inactive state that is maintained through replication regardless of histone acetylation and DNA methylation status. The disruption of many chromatin-modifying proteins are coincident with various human diseases, including cancer [94]. Although it has long been known that changes in cellular heterochromatin content are associated with more aggressive cancers [77,95], a recent report shows a direct correlation between HP1 relocalization and invasive/metastatic breast cancer [96]. On the basis of recent findings that HP1 binds the H3 Lys9-methyl modification, it is possible that the altered HP1 localization may be directly linked to the methylation status of Lys9. Besides altering genomic stability, the increase or decrease of H3 Lys9 methylation and HP1 binding could have a number of effects on the progression of a metastatic phenotype, including the aberrant silencing of tumor suppressor genes and/or activation of oncogenes, respectively.

Conclusions and future directions In this review, we have used histone acetylation as the paradigm to discuss the functional and biological importance of histone methylation in different cellular processes. Past reports and recent findings suggest that the methylation of lysine residues in histone tails functions as a stable epigenetic mark to localize chromatin-associated proteins in order to direct specific chromatin-mediated events. Furthermore, methylation of different lysine residues on the same histone tail in vivo can be associated with opposing biological processes such as transcriptional upregulation and heterochromatin assembly leading to epigenetic silencing. We suspect that histone methylation will have

widespread and far reaching implications in numerous epigenetic phenomena including, but not limited to, imprinting, X-inactivation, differentiation, transposition and programmed DNA rearrangements. In vivo sites of histone methylation remain to be determined, especially for H2A and H2B, and as presented earlier, arginine methylation has yet to be documented in cellular histones. Further studies are required to determine the biological significance of each modified residue. To this end it will be useful to generate an arsenal of antibodies specific for each known methylated residue in the histone tails, arginine or lysine. The use of these antibodies in ChIP experiments, in combination with high density microarrays of human gene promoters, would yield considerable insights into the global effects and mechanisms of histone methylation and transcriptional regulation in normal and diseased cells. In addition, it is likely that many more HMTs have yet to be discovered, each with their own substrate and site specificity. The discovery of new HMTs will probably result in the identification of novel conserved families of proteins that contain unique catalytic domains distinct from the well defined SET domain. In keeping with the flurry of research in the histone acetylation field during the past six years, ignited by the discovery of the transcription-associated HATs and HDACs [97,98], we anticipate that the discovery of the first HMTs [27••,29••] will lead to many more exciting discoveries centered around histone methylation. In fact, we predict that the methyl modification of histones may prove to be as significant in the regulation of chromatin structure and function as histone acetylation. Only time will tell.

Update A recent article by Nakayama et al. [99•] demonstrates the enzymatic methylation of H3 Lys9 is required for proper heterochromatin assembly in vivo. Using H3 Lys9-methyl specific antibodies in ChIP assays and fission yeast as the model system, it was shown that functional Clr4 was required for H3 Lys9 methylation, appropriate localization of Swi6 to the mat and cen loci, heterochromatin formation and epigenetic silencing. Furthermore, it was shown that other histone modifications, such as acetylation of H3 Lys9 and Lys14, negatively regulate this process. These findings provide novel insights into a conserved pathway of heterochromatin assembly, whereby sequential histone modifications lead to epigenetic silencing in organisms ranging from yeast to humans (Figure 3).

Acknowledgements In some cases, readers are directed to excellent reviews wherein many of the key primary literature are referenced. We apologize for not being able to cite all of the primary literature due to space limitations. We wish to thank Patrick Grant and current laboratory members for their input, especially Jim Bone, Peter Cheung and Craig Mizzen. In addition, we would also like to thank Upstate Biotechnology for their continued support. Research support to CDA is provided by a MERIT Award from the National Institutes of Health.

Histone methylation versus histone acetylation Rice and Allis

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