Histone methyltransferases

Histone-lysine methyltransferases are chromatin-bound enzymes that catalyses the addition of methyl groups onto lysine or arginine residues of chromatin-bound H3 and H4 [151]. The methyl group is transferred enzymatically to the histone with S-adenosyl methionine as the methyl donor. Histone methylases have been isolated from HeLa S-3 cells [182], chick embryo nuclei [183], and rat brain chromatin [184]. The histone methyltransferases methylated H3 and H4 in nucleosomes [184]. Histone-lysine methyltransferase is a chromatin-bound enzyme [129,151]. Initial characterization of the Tetrahymena macronuclear H3 methyltransferase suggests that the enzyme has a molecular mass of 400 kDa. The enzyme preferred free histones rather than nucleosomes as substrate [138]. More recent studies have now 

During the past several years, a variety of crystal structures of histone lysine and arginine methyltransferase in complex with the cofactor analog SAH and/or in complex with peptide substrates have been reported [92]. However, no 3D structure of a complex between a histone methyltransferase (HMT) and an inhibitor has been reported so far. Due to the lack of experimental structures, a variety of molecular modeling and docking studies has been carried out for H MTs in order to understand the structural requirements for inhibitor binding. 

We thank Barbara Eisner, Institute of Pharmacy, Martin-Luther-University of Halle-Wittenberg, for technical assistance. 

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2 Kouzarides, T. (2007) Chromatin modifications and their function. Cell, 128, 693-705. 

3 Finnin, M.S., Donigian, J.R., Cohen, A., Richon, V.M., Rrfkind, R.A., Marks, P.A. et al, (1999) Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature, 401, 188-193. 

Methylation of histone lysine residues is catalysed by site- and methylation-state specific i.e. tri-/di-/monomethylation states) histone methyltransferases (EC number 2.1.1.43, Table 5.4). With the exception of DOTIL, which methylates H3K79, all identified KMTs catalyse methylation using a SET domain (SET is an abbreviation for Su(var)3-9, Enhancer of zeste and Trithorax and refers to the Drosophila protein suppressor of variegation where this domain was first identified).  

Arginine methylation is catalysed by protein arginine methyltransferases (PRMTs, EC number 2.1.1.125), and thus far eight human PRMTs have been identified, with several more putative enzymes predicted. Two subclasses of PRMTs exist Class I PRMTs catalyse asymmetric dimethylation, while Class II PRMTs catalyse symmetric dimethylation of arginine. All three methyl-transferase subfamilies (SET-domain KMTs, DOTIL and PRMTs) utilise S-adenosylmethionine as an electrophilic methyl source. 

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Min J, Feng Q, Li Z, Zhang Y, Xu R (2003) Structure of the catalytic domain of human dotll, a non-set domain nucleosomal histone methyltransferase. Cell 112 711—723... 

Wilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackburn GM, Gamblin SJ, Xiao B (2002) Crystal structure and functional analysis of the histone methyltransferase set7/9. Cell 111 105-115... 

Xiao B, Jing C, Wilson JR, Walker PA, Vasisht N, Kelly G, Howell S, Taylor IA, Blackburn GM, Gamblin SJ (2003) Structure and catalytic mechanism of the human histone methyltransferase set7/9. Nature 421 652-656... 

Efforts to identify specific inhibitors of histone methyltransferases are in their early stages, and only two specific KMT inhibitors have been identified. The fungal mycotoxin chaetocin (9, Figure 2) inhibits the KMT Su(var)3-9 from Drosophila with an IC50 of 0.8 iM and produces a... 

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Table 2. List of known histone methyltransferases (HMTs)...

Hamamoto R, Eurukawa Y, Morita M, limura Y, Silva EP, Li M, Yagyu R, Nakamura Y (2004) SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol 8 731-740... 

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Gray SG, Ekstrom TJ (2001) The human histone deacetylase family. Exp Cell Res 262 75—83 Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A (2005) Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nat Chem BioH 143-145 Grozinger CM, Chao ED, Blackwell HE, Moazed D, Schreiber SL (2001) Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 276(42) 38837-38843... 

The mammalian inactive X-chromosomes in females is associated with H3 methylated at Lys-9 but does not have H3 methylated at Lys-4 [165,166]. Deletion of murine Suv39h genes does not affect H3 Lys-9 methylation at the inactive X-chromosome. Thus, different histone methyltransferases are involved in the methylation of H3 Lys-9 in constitutive and facultative heterochromatin [166]. Further, HPl is not associated with the inactive X-chromosome. Methylation of H3 Lys-9 is an early event in X inactivation in mammals [167,168]. The H3 Lys-9 methylation occurs approximately at the same time as H3 Lys-9 hypoacetylation and Lys-4 hypomethylation, and happens before transcriptional inactivation of the X-linked genes. Upstream of the Xist gene, which codes for a strictly nuclear RNA involved in X inactivation, is a constitutive hotspot of H3 methylated at Lys-9. This early event of H3 Lys-9 methylation occurs simultaneously with or immediately following the association of Xist RNA with the X-chromosome. It has been proposed that the hotspot upstream of the Xist gene serves as a nucleation site for the spreading of Xist RNA and methylated Lys-9 H3 [167]. 

Mammalian G9a is a SET-domain histone methyltransferase that methylated H3 at Lys-9 and Lys-27 in vitro and at Lys-9 in vivo [196]. The consensus sequence for G9a appears to be TKXXARKS. G9a is dilferent from Suv39hl is several ways. G9a nuclear localization is distinct from that of Suv39hl, which locate to heterochromatic foci [197]. Suv39hlj2 double mutant mice lose H3 Lys-9 methylation at pericentromeric heterochromatic regions but broad methylation of chromatin remains. It is the latter that is lost in GPa-deficient cells [196]. G9a, molecular mass about 100 kDa, methylates free H3 and nucleosomal H3 with a preference for the former however, the presence of H1 stimulates the methylation of chromatin substrates. Suv39hl, molecular mass about 650 kDa, methylates free H3 and H3 in nucleosomes with equivalent efficiency, but when HI is present, methylation of chromatin substrates is lessened [198]. 

Human Set9 is a 50 kDa H3 methyltransferase that methylates Lys-4 of H3. The enzyme methylated free H3 but not H3 in chromatin substrates. There is evidence that Set9 may stimulate activated transcription [198]. Set9 has the SET domain but lacks the cysteine-rich (pre-SET and post-SET) domains. Disruption of Saccharomyces cerevisiae and Saccharomyces pombe Setl obliterates H3 methyl Lys-4 [199]. Thus this SET domain containing protein appears to be a H3 Lys-4 methyltransferase, catalyzing both di- and tri-methylation of H3 Lys-4 [155]. However, studies with recombinant Setl failed to show histone methyltransferase activity. It has been suggested that other associated proteins may be required for the Setl to be catalytically active [139,200]. Indeed, Setl is associated with several... 

Histone methyltransferases may associate or act cooperatively with either HATs or HDACs. In fission yeast the H3 Lys-14 deacetylase Clr3 interacts functionally with H3 Lys-9 methyltransferase Clr4. Clr4 methylates Lys-9 of H3, a process facilitated by Rikl, resulting in the recruitment of Swi6 and heterochromatin assembly [157,221]. In Drosophila SU(VAR)3-9 H3 Lys-9 methyltransferase is in complex with HDACl [222]. Thus, HDACl would deacetylate acetylated Lys-9 allowing methylation by SU(VAR)3-9 at this site to occur. CBP, a potent HAT, is associated with a histone methyltransferase that methylated H3 at Lys-9 and to a lesser extent Lys-4. H3 methylation at Lys-9 did not alter the HAT activity of CBP, and vice versa acetylation of H3 (predominantly Lys-14) did not affect the associated histone methyltransferase activity [223]. 

Horita, D.A., Ivanova, A.V., Altieri, A.S., Klar, A.J., and Byrd, R.A. (2001) Solution structure, domain features and structural implications of mutants of the chromo domain from the fission yeast histone methyltransferase Clr4. J. Mol. Biol. 307, 861-870. 

In fact, the cross-talk between histone methylation and DNA methylation has been experimentally demonstrated for the first time in Neurospora [145] (see commentary by Bird [146]) and proved the second scenario. Using the power of Neurospora genetics, Tamaru and Selker [145] have identified a gene which, when mutated, abolishes methylation of all tested DNA sequences. This gene, dim-5, is different from dim-2, the previously identified gene that encodes the only DNA methyltransferase in Neurospora responsible for all known cytosine methylation in this fungus. The dim-5 gene encodes a histone H3 methyltransferase the deduced polypeptide sequence contains a SET domain with sequence similarity to some known histone methyltransferases moreover, the recombinant DIM-5 protein exhibits histone methyltransferase activity in vitro. Additional in vivo experiments... 

Fig. 8. Proposed models that link histone methylation to DNA methylation (for details see Section 5.2). Methylated cytosines attract histone methyltransferases that contain a methyl-binding domain or a methyl-CpG binding protein (MeCP2) that recruits histone methylase activities these introduce methyl groups into the histone tails. The binding of chromodomain HPl proteins to H3 tails methylated at lysine 9 generates a secondary layer of repressive chromatin structure, (b) In a reverse scenario, methylated histone tails attract chromodomain-binding proteins, which in turn recruit Dmnts to methylate adjacent DNA sequences.

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