MINIREVIEW Mixed lineage leukemia: histone H3 lysine 4 methyltransferases from yeast to human Shivani Malik and Sukesh R. Bhaumik Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL, USA Introduction The DNA in eukaryotes is compacted in the form of chromatin. The fundamental unit of chromatin is the nucleosome which consists of a core histone particle with 146 bp of DNA wrapped around it [1,2]. The core histone particle comprises a tetramer of histones H3 and H4 and dimers of histones H2A and H2B [2]. Each of these histones has a structured core globular domain and an unstructured flexible N-terminal tail protruding from the core domain. The linker histone H1 associates with the core domain to form a higher order structure, thus further compacting the DNA [3,4]. Such compac- tion of DNA in a higher order chromatin structure makes it inaccessible for proteins involved in different DNA-transacting processes such as transcription, repli- cation, recombination and DNA repair. However, the chromatin structure has to be dynamic in nature in order for DNA-transacting processes to occur [5–10], and such dynamic states are regulated by ATP-depen- dent chromatin remodelers as well as by ATP-indepen- dent histone covalent modifications. There are several ATP-dependent chromatin remo- delers. These include the switching–defective ⁄ sucrose non-fermenting (SWI ⁄ SNF), imitation switch (ISW1), nucleosome remodeling and histone deacetylation (Mi-2 ⁄ NuRD), and INO80 complexes [11–25]. These complexes have a catalytic ATPase subunit with Keywords ASH1; ASH2; COMPASS; histone H3 lysine 4; histone methyltransferase; MLL; Set1; TAC1; TRX; WDR5 Correspondence S. R. Bhaumik, Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA Fax: +1 618 453 6440 Tel: +1 618 453 6479 E-mail: sbhaumik@siumed.edu (Received 16 November 2009, revised 12 January 2010, accepted 22 January 2010) doi:10.1111/j.1742-4658.2010.07607.x The fourth lysine of histone H3 is post-translationally modified by a methyl group via the action of histone methyltransferase, and such a covalent modification is associated with transcriptionally active and ⁄ or repressed chromatin states. Thus, histone H3 lysine 4 methylation has a crucial role in maintaining normal cellular functions. In fact, misregulation of this covalent modification has been implicated in various types of cancer and other diseases. Therefore, a large number of studies over recent years have been directed towards histone H3 lysine 4 methylation and the enzymes involved in this covalent modification in eukaryotes ranging from yeast to human. These studies revealed a set of histone H3 lysine 4 methyltransfe- rases with important cellular functions in different eukaryotes, as discussed here. Abbreviations ASH1, absent, small or homeotic discs 1; ASH2, absent, small or homeotic discs 2; BRM, brahma; CBP, CREB-binding protein; EcR, ecdysone receptor; HAT, histone acetyl transferase; H3K4, histone H3 lysine 4; HMT, histone methyltransferase; MLL, mixed lineage leukemia; MOF, male absent on the first; Paf1, RNA polymerase II-associated factor 1; PcG, polycomb group; PHD, plant homeodomain; TAC1, trithorax acetylation complex 1; TRR, trithorax-related; TRX, trithorax. FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS 1805 DNA-dependent ATPase activity. ATP-independent histone covalent modifications are acetylation, phos- phorylation, ubiquitylation, methylation, sumoylation and ADP ribosylation [8,10,26–33]. Although most of these modifications occur on the N-terminal tails of histones, some also occur on the C-terminal tails of histones H2A and H2B [29,30,34] and the core region of histone H3 [30,31,35,36]. These covalent modifica- tions have profound effects on chromatin structure and hence gene regulation [5,8,9,30,33]. H3 N-A R T K R K S T K R K K R K S K C . . K 4 9 14 18 23 27 36 79 me A B me me me me Set1 (Sc) Set1 (Sp) SET1 (Ce) TRX (Dm) ASH2 (Dm) TRR (Dm) ASH1 (Dm) ASH1 (Hs) MLL 1-4 (Hs) SET1A (Hs) SET1B (Hs) SMYD3 (Hs) SET7/9 (Hs) Meisetz (Hs) Clr4 (Sp) SU(VAR)3-9 (Dm) G9a (Dm) ASH1 (Dm) Eu-HMTase 1 (Hs) SUV39 H1 (Hs) SUV39 H2 (Hs) G9a (Hs) ESET or SETDB1 (Hs) MES-2 (Ce) E (Z) (Dm) G9a (Dm) EZH2 (Hs) EZH1 (Hs) G9a (Hs) Set2 (Sc) Set2 (Sp) Set2 (Dm) ASH1 (Dm) NSD1 (Hs) SETD2/HYPB (Hs) Dot1 (Sc) Dot1 (Sp) DOT1L (Hs) Gene activation, telomeric silencing & DNA repair Hetero- chromatin formation & silencing Hetero- chromatin formation & silencing Inhibition of intragenic transcription & hetero- chromatin spreading, & regulation of transcriptional elongation Gene activation & silencing, splicing, & DNA repair Lysine methylases Functions Lysine methylases Functions H4 N-SGRGKGGKGLGKGGAKRHRKV C 5 8 12 16 20 me Heterochromatin formation & silencing Set9 (Sp) Pr-SET7 (Dm) ASH1 (Dm) SUV4-20 (Hs) Pr-SET7/SET8 (Hs) Fig. 1. Methylation of different lysine (K) residues of histones H3 (A) and H4 (B) with associated methylases and functions in genome expression and integrity. Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens. H3K4 methyltransferases from yeast to human S. Malik and S. R. Bhaumik 1806 FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS The lysine (K) residues of histones H3 and H4 can be mono-, di- and trimethylated, and such methylation is associated with active and ⁄ or repressed chromatin (Fig. 1). Thus, histone methylation is linked to diverse cellular regulatory functions [27,30,31,33]. Indeed, sev- eral studies have implicated histone methylation in var- ious types of cancer and other diseases [30,33,37–39]. Therefore, a large number of studies over several years have focused on histone methylation at different K residues and the enzymes involved in this covalent modification in diverse eukaryotes [27,30,31,33,40–45]. These studies have revealed several histone meth- yltransferases (HMTs) involved in the K methylation of histones H3 and H4 with crucial roles in maintain- ing normal cellular functions in eukaryotes ranging from yeast to humans (Fig. 1). Here, we discuss his- tone H3 lysine 4 (H3K4) methylation and the HMTs involved in this covalent modification, highlighting the similarities and differences in several eukaryotes such as Saccharomyces cerevisiae (budding yeast), Schizosac- charomyces pombe (fission yeast), Caenorhabditis elegans (roundworm), Drosophila melanogaster (fruit fly), Mus musculus (mouse) and Homo sapiens (human). H3K4 methylation and HMTs in Saccharomyces cerevisiae In S. cerevisiae, H3K4 methylation is involved in the stimulation of transcription [29–31,33]. Further, H3K4 methylation in S. cerevisiae has been implicated in silencing at telomeres, ribosomal DNA and the mat- ing-type locus [30,33,46,47]. Thus, H3K4 methylation participates in both gene activation and repression. The enzyme responsible for this covalent modification was first identified in a multiprotein complex, named COMPASS, in S. cerevisiae [48]. COMPASS consists of the catalytic subunit, Set1, and seven other proteins (Cps60 ⁄ Bre2, Cps50 ⁄ Swd1, Cps40 ⁄ Spp1, Cps35 ⁄ Swd2, Cps30 ⁄ Swd3, Cps25⁄ Sdc1 and Cps15 ⁄ Shg1) (Tables 1–3) [29,33,48,49]. Set1 is essential for mono-, di- and trimethylation of histone H3 at K4 [29,30,33, 48,49]. Set1 is enzymatically active only when assem- bled into the multi-subunit COMPASS complex. The ability of COMPASS to mono-, di- and trimethylate K4 of histone H3 depends on its subunit composition. For example, COMPASS lacking Cps60 ⁄ Bre2 cannot trimethylate K4 of histone H3, whereas the Cps25 subunit of COMPASS is essential for histone H3 K4 di- and trimethylation [29,33,49,50]. The COMPASS complex preferentially associates with RNA polymer- ase II which is phosphorylated at Ser 5 in its C-termi- nal domain at the onset of transcriptional elongation [33,49,51–54]. The interaction between COMPASS and RNA polymerase II is further facilitated by the RNA polymerase II-associated factor 1 (Paf1) complex which associates with the coding sequence in an RNA polymerase II-dependent manner during transcriptional elongation [33,49,51–54]. Thus, COMPASS is found to be predominantly associated with the coding sequences of active genes [33,49,51,54,55], and the coding sequences of the actively transcribing genes are there- fore trimethylated at the K4 of histone H3 [33,51,54– 58]. Interestingly, the methyltransferase activity of the COMPASS complex is intimately regulated by ubiqui- tylation of histone H2B at K123 [30,33,59–63]. Both di- and trimethylation of histone H3 at K4 are impaired in the absence of histone H2B K123 ubiqui- tylation, which is catalyzed by E2 ubiquitin conjugase and E3 ubiquitin ligase, Rad26 and Bre1, respectively. However, histone H2B K123 ubiquitylation does not regulate H3K4 monomethylation [33,62,64]. Such a trans-tail cross-talk between histone H2B K123 ubiquitylation and H3K4 di- and trimethylation is mediated via alteration of the subunit composition of COMPASS [33,55]. It was recently demonstrated that histone H2B K123 ubiquitylation is essential for the recruitment of Cps35 ⁄ Swd2 independent of Set1 [33,55]. Set1 maintains the structural integrity of the COMPASS complex [33,55]. COMPASS without Table 1. The histone H3 lysine 4 methyltransferases in different eukaryotes (references are cited in the text). Saccharomyces cerevisiae Schizosaccharomyces pombe Caenorhabditis elegans Drosophila Mammals (mouse and human) COMPASS ⁄ Set1C Set1C COMPASS-like complex TAC1 ASH1 ASH2 TRR MLL1 MLL2 MLL3 MLL4 SET1A SET1B SET7 ⁄ 9 SMYD3 ASH1 Meisetz S. Malik and S. R. Bhaumik H3K4 methyltransferases from yeast to human FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS 1807 Cps35 ⁄ Swd2 is consistently recruited to the coding sequence of the active gene in an RNA polymerase II- dependent manner in the absence of histone H2B K123 ubiquitylation [33,55]. COMPASS without Cps35 ⁄ Swd2 monomethylates K4 of histone H3, but does not have catalytic activity for di- and trimethyla- tion of histone H3 at K4 [33,55]. When histone H2B is ubiquitylated by the combined actions of Rad26 and Bre1, it recruits Cps35 ⁄ Swd2, which interacts with the rest of COMPASS recruited by elongating RNA poly- merase II. Such interaction leads to the formation of a fully active COMPASS capable of H3K4 mono-, di- and trimethylation [33,55]. Thus, H3K4 methylation is regulated by upstream factors involved in histone H2B K123 ubiquitylation. Further, H3K4 methylation is controlled by a demethylase with the Jumonji C (JmjC) domain, namely Jhd2, which specifically deme- thylates the trimethylated K4 of histone H3 (Table 4) [65]. Such demethylation provides an additional level of regulation of H3K4 methylation in S. cerevisae. H3K4 methylation and HMTs in Schizosaccharomyces pombe Although the first H3K4 methyltransferase was identified in S. cerevisiae, the chromatin structure in S. cerevisiae is not similar to that of Sch. pombe or higher eukaryotes. For example, S. cerevisiae lacks Table 2. The components of characterized histone H3 lysine 4 methyltransferase complexes in different eukaryotes (references are cited in the text). Saccharomyces cerevisae Schizosaccharomyces pombe Caenorhabditis elegans Drosophila Mammals COMPASS ⁄ Set1C Set1 Cps60 ⁄ Bre2 Cps50 ⁄ Swd1 Cps40 ⁄ Spp1 Cps35 ⁄ Swd2 Cps30 ⁄ Swd3 Cps25 ⁄ Sdc1 Cps15 ⁄ Shg1 Set1C Set1 Ash2 Swd1 Spp1 Swd2 Swd3 Sdc1 Shg1 COMPASS-like complex SET-2 ⁄ SET1 ASH2 ⁄ Y17G7B.2 CFPL-1 SWD-3 SWD-2 ⁄ C33H5.6 SPP-1 ⁄ F52B11.1 DPY-30 TAC1 TRX CBP SBF1 ASH1 ASH1 ? ASH2 ASH2 ? TRR TRR ? MLL1 MLL1 ASH2L WDR5 RBBP5 DPY-30 HCF1 ⁄ HCF2 Menin MOF MLL2 MLL1 ASH2L WDR5 RBBP5 DPY-30 Menin HCF2 RPB2 MLL3 ⁄ MLL4 MLL3 ⁄ MLL4 ASH2L WDR5 RBBP5 DPY-30 NCOA6 PA1 PTIP UTX SET1A ⁄ SET1B SET1A ⁄ SET1B ASH2L WDR5 RBBP5 WDR82 ⁄ SWD2 a CFP1 ⁄ CGBP a DPY-30 HCF1 a Wdr82 and CFP1 ⁄ CGBP are present in human SET1A and SET1B complexes, but not in mouse [117,165,166]. H3K4 methyltransferases from yeast to human S. Malik and S. R. Bhaumik 1808 FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS homologs of the repressive histone H3K9 methyltransfe- rases and the heterochromatin proteins (e.g. HP1) present in Sch. pombe or higher eukaryotes [66–68]. Thus, Sch. pombe serves as a better eukaryotic model system to study the roles of H3K4 methylation in regulation of chromatin structure and gene expression. As in S. cerevisae, H3K4 methylation in Sch. pombe is catalyzed by a SET domain-containing protein, Set1. The Sch. pombe Set1 protein is homologous to S. cerevi- siae Set1. Set1 proteins in budding and fission yeasts share a high degree of similarity in their SET domains. However, these two proteins exhibit 26% sequence iden- tity overall [69]. The N-termini of Set1 in S. cerevisae and Sch. pombe are considerably different [69–71]. Such difference might have crucial roles in governing the specific functions of Set1 in S. cerevisae and Sch. pombe. For example, a recombinant Sch. pombe Set1 methylates K4 in a 20-amino acid peptide corresponding to the N-terminal tail of histone H3 in vitro. By contrast, recombinant S. cerevisae Set1 does not have methyl- transferase activity in vitro. Further, phylogenetic analy- sis indicates that Sch. pombe Set1 is more closely related to human Set1 than to S. cerevisae Set1 [69]. Sch. pombe Set1 mutants have slow growth, exhibit temperature- sensitive growth defects and have a slightly longer dou- bling time compared with wild-type cells [69]. DNA sequence analysis reveals that homologs of the components of S. cerevisae COMPASS are also pres- ent in Sch. pombe. Indeed, Set1 methyltransferase com- plex (Set1C) has been purified in Sch. pombe, which shares many features of S. cerevisae COMPASS (Tables 1–3). However, these two complexes differ in several ways. For example, the Ash2 component of Set1C in Sch. pombe has a plant homeodomain (PHD) finger domain, whereas the homologous protein, Cps60 ⁄ Bre2 (Table 3), in S. cerevisae does not [70]. The Cps40 ⁄ Spp1 component (that bears the PHD fin- ger domain) is required for methylation in Sch. pombe, but not in S. cerevisae [70]. Furthermore, Set1C in Sch. pombe shows a hyperlink to Lid2C (little imaginal discs 2 complex) through Ash2 and Sdc1 [70]. How- ever, such a hyperlink is absent in S. cerevisae.In addition, the identified hyperlink, Swd2 (which is also a subunit of the cleavage and polyadenylation factor) in S. cerevisae COMPASS is not found in Sch. pombe [70–72]. Together, these observations support the fact that the Set1 HMTs from S. cerevisae and Sch. pombe are highly conserved (Tables 1–3), but their proteomic environments appear to differ. However, such differ- ences in the proteomic environments may be related to the absence of histone H3 K9 methylation in S. cerevi- siae, as suggested previously [70]. Table 4. Histone H3 lysine 4 demethylases in different eukaryotes (references are cited in the text). me 3 , trimethyl; me 2 , dimethyl; me 3 ⁄ 2 , tri- and dimethyl; and me 2 ⁄ 1 , di- and monomethyl. Saccharomyces cerevisae Schizosaccharomyces pombe Caenorhabditis elegans Drosophila Mammals (mouse and human) Jhd2 (me 3 ) Swm1 (me 2 ) Swm2 (me 2 ) SPR-5 (me 2 ) T08D10.2 (me 2 ) R13G10.2 (me 2 ) Lid (me 3 ) LSD1 (me 2 ⁄ 1 ) JARID1A (me 3 ⁄ 2 ) JARID1B (me 3 ⁄ 2 ) JARID1C (me 3 ⁄ 2 ) JARID1D (me 3 ⁄ 2 ) Table 3. Homologous subunits of histone H3 lysine 4 methyltransferase complexes in different eukaryotes (references are cited in the text). Saccharomyces cerevisiae Schizosaccharomyces pombe Caenorhabditis elegans Drosophila Mammals Set1 Set1 SET-2 ⁄ SET1 TRX MLL1-4, SET1A ⁄ SET1B Cps60 ⁄ Bre2 Ash2 ASH-2 ASH2 ASH2L ⁄ ASH2 Cps50 ⁄ Swd1 Swd1 CFPL-1 RBBP5 Cps30 ⁄ Swd3 Swd3 SWD-3 WDR5 WDR5 Cps35 ⁄ Swd2 Swd2 SWD-2 WDR82 ⁄ SWD2 Cps40 ⁄ Spp1 Spp1 SPP-1 CFP1 Cps25 ⁄ Sdc1 Sdc1 DPY-30 DPY-30 Cps15 ⁄ Shg1 Shg1 ASH1 ASH1L ⁄ ASH1 TRR Menin; HCF1 ⁄ HCF2; NCOA6; PA1; PTIP; UTX; MOF; SET7 ⁄ 9; SMYD3; Meisetz S. Malik and S. R. Bhaumik H3K4 methyltransferases from yeast to human FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS 1809 H3K4 methylation is correlated with active chroma- tin in Sch. pombe [69], as in S. cerevisae. However, unlike in S. cerevisiae, H3K4 methylation is not required for silencing and heterochromatin assembly at the centromeres and mating type locus in Sch. pombe [69], possibly because of the presence of repressive histone H3 K9 methylation in Sch. pombe [69]. Fur- thermore, previous studies [69] have demonstrated that H3K4 methylation is correlated with histone H3 acety- lation in Sch. pombe, and hence is associated with active genes. However, transcriptional stimulation by H3K4 methylation is also closely regulated by histone demethylase, LSD1, which is absent in S. cerevisae. LSD1 is an amine oxidase which demethylates the K residue in a FAD-dependent manner. Because it functions through oxidation, it can only demethylate mono- and dimethylated K4 of histone H3. There are two LSD1-like proteins, namely Swm1 and Swm2 (after SWIRM1 and SWIRM2), in Sch. pombe (Table 4) [73,74] which form a complex and are involved in the demethylation of methylated K4 and K9 of histone H3. Such a demethylation process has important roles in the regulation of chromatin struc- ture and hence gene expression. H3K4 methylation and HMTs in Caenorhabditis elegans C. elegans is a multicellular, yet simple, eukaryotic sys- tem with technical advantages for studying the chro- matin structure in greater detail. Thus, C. elegans can serve as a model system to understand the role of histone covalent modifications in developmental pro- cesses. As in S. cerevisae and Sch. pombe, H3K4 meth- ylation has an important role in promoting transcription in C. elegans [75]. However, early C. ele- gans embryos have a transcriptionally repressed chro- matin state, even though both di- and trimethylation of histone H3 at K4 are present in the chromatin of the germline blastomere [75]. Such repression perhaps results from the lack of Ser 2 phosphorylation in the C-terminal domain of the largest RNA polymerase II subunit in the germline cells [76]. Following division of the germline lineage P4 cells into the primordial germ cells, H3K4 methylation is lost [75]. However, H3K4 methylation is regained prior to postembryonic prolif- eration. Such covalent modification activates gene expression in the postembryonic germ cells [75,77]. The enzyme involved in H3K4 methylation in C. ele- gans was identified recently via an RNAi screen of the suppressors of heterochromatin protein mutants (hpl-1 and hpl-2). The RNAi screen identified set-2 as the homolog of yeast SET1 [78]. Further, several studies have revealed that SET-2 (also known as SET- 2 ⁄ SET1) forms a complex with SWD-3, CFPL-1, DPY-30, Y17G7B.2 ⁄ ASH-2, C33H5.6 ⁄ WDR82 ⁄ SWD- 2 and F52B11.1 ⁄ SPP-1 (Tables 1 and 2) [78]. These proteins are homologous to the budding yeast COM- PASS (Table 3). Thus, as in S. cerevisae, SET-2 ⁄ SET1 in C. elegans forms a COMPASS-like complex. Fur- thermore, like in fission yeast, SET-2 ⁄ SET1 may be hyperlinked to the complex that is homologus to Sch. pombe’s Lid2C-containing Sdc1. In support of this notion, DPY-30 in C. elegans was found to be the homolog of fission yeast Sdc1 (Table 3), and it plays an important role in dosage compensation [79]. Thus, it is likely that SET-2 ⁄ SET1 in C. elegans is connected to another complex via DPY-30, which remains to be elucidated. As in yeast, the subunits of the COMPASS-like complex in C. elegans differentially regulate global H3K4 methylation [78]. For example, no decrease in the global level of H3K4 methylation was observed upon RNAi-based depletion of swd-2 [78]. A moderate decrease in H3K4 methylation was observed in the absence of Y17G7B.2 ⁄ ASH-2. Depletion of set-2, swd-3, cfpl-1 and dpy-30 led to a drastic decrease in global H3K4 methylation, with the most severe defect observed in swd-3 mutants. However, unlike in yeast, the residual level of global H3K4 methylation was observed in the absence of SET-2 ⁄ SET1 activity [78]. This observation suggests that additional H3K4 methyltransferase(s) may exist in C. elegans. Although H3K4 methylation is associated with active transcription in C. elegans, it is reset during gametogenesis. A demethylase, SPR-5, has recently been identified in C. elgans, and it shows 45% similar- ity with human LSD1 [80]. SPR-5 is responsible for demethylation of dimethylated K4 of histone H3 (Table 4). It interacts with the (co)repressor for ele- ment-1-silencing transcription factor repressor protein, SPR-1 [81–83]. Such an interaction is correlated with the repressive role of SPR-5 in gene regulation. Like SPR-5, two other proteins in C. elegans, namely T08D10.2 and R13G10.2, have a LSD1-like amine oxi- dase domain, as revealed by the NCBI Conserved Domain Search Program (Table 4) [84]. Knockdown of T08D10.2 by RNAi extends the longevity, thus implicating the role of histone methyaltion in regula- tion of aging [85]. Although studies in C. elegans have been quite help- ful in understanding the role of H3K4 methylation in gene expression and development, the pattern of cell lineage in C. elegans is highly invariant [86]. However, the development of embryos of Drosophila and mam- mals largely relies on cellular cues, thus making it a H3K4 methyltransferases from yeast to human S. Malik and S. R. Bhaumik 1810 FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS more complex process. Therefore, studies in Drosophila will provide a better understanding of the regulatory roles of H3K4 methylation in gene expression and development. H3K4 methylation and HMTs in Drosophila Drosophila has long been a model organism for studying developmental processes, because development in humans and Drosophila are homologous processes. Dro- sophila and humans share a number of related develop- mental genes working in conserved pathways. Studies analyzing the interplay of the SET domain-containing trithorax group (trxG) and polycomb group (PcG) pro- teins in regulating transcription patterns during develop- ment and differentiation have been an area of extensive research in Drosophila [87–91]. PcG and trxG proteins play a crucial role in the epigenetic control of a large number of developmental genes, including the Hox (Homeotic) genes [92–99]. Hox are a cluster of genes that defines the anterior–posterior axis and segment identity during early embryogenesis. The expression pat- tern of the Hox genes is established early in development and is propagated in appropriate cell lineages [100]. However, transcription of Hox genes is closely regulated by the antagonistic actions of PcG and trxG proteins through different patterns of histone methylation. The PcG protein, E(Z), methylates histone H3 at K27 [100]. Further, histone H3 methylated at K27 is recognized by a chromodomain-containing PcG protein, PC [100]. These events repress Hox and other target genes. How- ever, the trxG proteins such as trithorax (TRX) and absent, small or homeotic discs 1 (ASH1) are H3K4 methyltransferases which promote transcription of the target genes including Hox [100]. Thus, a close interplay between the specific PcG and trxG proteins maintains a tight regulation of Hox gene expression. Consistently, genetic studies in Drosophila have revealed that muta- tions in specific PcG and trxG genes result in flies with homeotic transformations because of the misregulation of Hox genes [100–103]. As mentioned above, TRX is a member of the trxG proteins with H3K4 methyltransferase activity in Drosophila, and it contains SET and PHD finger domains. TRX is the homolog of yeast Set1 in Dro- sophila, and is an integral component of a 1 MDa complex, called trithorax acetylation complex 1 (TAC1). TAC1 consists of TRX, CREB-binding pro- tein (CBP) and an anti-phosphatase SBF1 (Tables 2 and 3) [104]. Like mammalian CBP, Drosophila CBP has histone acetyl transferase (HAT) activity [104]. Thus, TAC1 possesses both HMT and HAT activities [94,104] which are associated with active transcription. The components of TAC1 are found to be associated with specific sites on salivary gland polytene chromo- somes, including Hox genes [104], and thus exist together in vivo. Mutations in either trx or the gene encoding CBP reduce the expression of a Hox gene, namely Ultrabithorax (Ubx) [104]. Thus, the two differ- ent enzymatic activities of TAC1 are closely linked to Hox gene expression [104]. Moreover, the HAT activ- ity of TAC1 may be counteracted by the deacetylase activity of the PcG complex, ESC ⁄ E(Z), accounting in part for the antagonistic functions of the trxG and PcG protein complexes on chromatin. Unlike its role in the regulation of Hox gene expression, TAC1 also promotes transcription of heat shock genes in a differ- ent mechanism through activation of poised or stalled RNA polymerase II. Heat shock genes are rapidly expressed by heat shock factor and other transcription factors [94,105]. TAC1 is recruited to several heat shock gene loci following heat induction, and conse- quently, its components are required for heat shock gene expression [94,105]. Smith et al. [94] demonstrate that TAC1 associates with transcription-competent stalled RNA polymerase II at the heat shock gene, and subsequently modifies histone H3 by methylation and acetylation. Such modifications of histone H3 facilitate stalled RNA polymerase II to begin transcriptional elongation [94,105]. In contrast to the results at heat shock genes, poised or stalled RNA polymerase II is not found at the Hox genes [94,105]. Thus, TAC1 appears to regulate the transcription of Hox and heat shock genes in distinct pathways. In addition to TRX, two other trxG proteins, namely ASH1 and ASH2, methylate K4 of histone H3 [106–110]. Mutations in the Ash1 and Ash2 genes gen- erate abnormal imaginal discs in flies [106–110], consis- tent with their roles in the regulation of Hox gene expression. Amorphic and antimorphic mutations in the Ash1 gene lead to a drastic decrease in the global level of H3K4 methyaltion [106–110]. The catalytic domain of ASH1 is 588 amino acids long, and com- prises the SET domain and cysteine-rich pre-SET and post-SET domains [106,107]. However, biochemical studies demonstrate that the 149-amino acid SET domain alone can methylate histone H3 at K4 in vitro [107]. ASH1 also contains a PHD finger, and a bromo-associated homology domain [111]. The bromo- associated homology domain of ASH1 might be responsible for protein–protein interactions during chromatin remodeling at the target genes. ASH1 is an integral component of a large 2 MDa complex [112]. In addition to H3K4 methylation, the ASH1 complex also methylates K9 of histone H3 and K20 of S. Malik and S. R. Bhaumik H3K4 methyltransferases from yeast to human FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS 1811 histone H4 [106,107]. Recently, Tanaka et al. [113] implicated ASH1 in methylation of histone H3 at K36. Apart from its role in histone methylation, ASH1 is also linked to histone acetylation via its interaction with CBP [114] which is an integral component of TAC1. Thus, ASH1 and TAC1 appear to have com- mon roles via CBP. Like ASH1, ASH2 is present in a 500 kDa complex [112]. ASH2 has been proposed to be the associated form of Bre2 and Spp1 of S. cerevisae COMPASS [48,71,115]. In mammals, ASH2 is a shared component of different complexes including a HMT bound by host cell factor 1 (HCF-1), Menin-containing complex and the COMPASS counterpart [116–120], indicating that it might be involved in the regulation of many dif- ferent processes. However, its role in histone methyla- tion is not known. Recently, Steward et al. [121] demonstrated that ASH2 in mammalian system has an important role in H3K4 trimethylation. Consistent with this observation, Beltran et al. [122] observed a severe reduction in H3K4 trimethylation in ash2 mutants. This observation indicates that ASH2 might play a crucial role in H3K4 methyltransferase activity. However, ASH2 does not contain a SET domain, but it has the PHD finger and SPRY domains [123]. In addition to its role in H3K4 methylation, ASH2 is also linked to histone deacetylation through its interaction with Sin3A, a histone deacetylase [116]. Further, ASH2 has been implicated in the regulation of cell- cycle progression via its interaction with HCF-1 [116]. Like trxG proteins, a trithorax-related (TRR) protein in Drosophila is also involved in the methylation of his- tone H3 at K4 [124]. TRR contains the SET domain, and has H3K4 methyltransferase activity [124]. TRR functions upstream of hedgehog (hh) in the progression of the morphogenic furrow [124]. It also participates in retinal differentiation [124]. TRR and trimethylated his- tone H3 at K4 are detected at the ecdysone-inducible promoters of hh and BR-C (broad complex) [124]. Ecdysone functions through binding to a nuclear recep- tor, ecdysone receptor (EcR), which heterodimerizes with the retinoid X receptor homolog ultraspiracle. The heterodimer is then recruited to the promoters of the target genes to regulate their expression, and hence ecdysone triggers molting and metamorphosis. Thus, the association of EcR along with TRR and H3K4 methylation is also observed at the hh and BR-C promoters following ecdysone treatment in cultured cells [124]. Consistent with these observations, H3K4 methylation is decreased at these promoters in embryos lacking functional TRR [124]. Thus, TRR appears to function as a coactivator at the ecdysone-responsive promoters by modulating the chromatin structure. H3K4 methylation functions as a platform for the binding of different chromatin remodelers. One such remodeler is the BRM complex which contains at least seven proteins [112]. Three components of the BRM complex are trxG proteins. These are BRM (brahma), Osa and Moira. However, these trxG protein compo- nents of the BRM complex do not have the SET domain as well as HMT activity. The BRM complex is the homolog of the yeast SWI ⁄ SNF complex, and shares four components including the ATPase BRM [112]. BRM also contains a high-mobility-group B pro- tein, namely BAP111, which binds nonspecifically to the minor groove of the double-helix and bends the DNA [125,126]. The BRM complex has ATP-depen- dent chromatin-remodeling activity. Mutations in ash1 enhance brm mutations, suggesting that they might be functioning together [110]. Consistent with this obser- vation, Beisel et al. [106] demonstrated that epigenetic activation of Ubx transcription coincides with H3K4 trimethylation by ASH1 and recruitment of the BRM complex. Similarly, mutations of ash2 and brm cause developmental defects in adult sensory organs includ- ing campaniform sensilla and mechanosensory bristles [108,127]. Thus, although ASH1, ASH2 and BRM are the components of three distinct complexes, they appear to function in concert to regulate transcription. Furthermore, the H3K4 methyltransferase activity of TAC1 has been implicated to be linked to the BRM complex [112,128]. Such linkage is mediated by the interaction of TRX of TAC1 with the SNR1 compo- nent of the BRM complex [128]. Together, these results indicate that H3K4 methylation and ATP- dependent chromatin remodeler, BRM, function in a concerted manner to regulate transcription. Apart from the BRM complex, two other chromatin remo- delers, namely nucleosome remodeling factor and ATP-utilizing chromatin assembly and remodeling fac- tor, have been implicated in transcriptional stimulation through their binding to methylated-K4 of histone H3 which is mediated by TRX or other HMTs [100]. Both nucleosome remodeling factor and ATP-utilizing chro- matin assembly and remodeling factor are ATP-depen- dent remodelers and carry ISW1 as an ATPase subunit [100]. Thus, the HMTs mark a modification pattern on histone H3 at K4 that is ‘read’ by chromatin remodel- ers which, in turn, regulate the chromatin structure and hence gene expression. Apart from H3K4 methyltransferase and ATP- dependent chromatin remodeling activities, trxG pro- tein is also involved in histone demethylation. Recent studies [129,130] have demonstrated that a trxG pro- tein, namely Lid, contains a JmjC domain and other functional domains found in mammalian Jumonji, H3K4 methyltransferases from yeast to human S. Malik and S. R. Bhaumik 1812 FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS AT-rich interactive domain 1 (JARID1) proteins. Lid has demethylase activity which can demethylate the trimethylated form of histone H3 at K4 (Table 4). Such demethylase activity of the trxG protein adds an additional layer to gene regulation by the PcG and trxG proteins. Further, Lid interacts with the proteins associated with heterochromatin formation such as H3 K9 methyltransferase [Su(var)3-9], heterochromatin protein (HP1) and deacetylase (RPD3). Thus, Lid plays crucial roles in removing the activation marks, hence facilitating gene silencing. The role of H3K4 methylation and its regulation in Drosophila is largely conserved in mammals. However, the complexity of mammals demands a more intricate mechanism of regulation in determining the cell lin- eages and developmental fates. Thus, a large number of studies have focused on H3K4 methylation and HMTs, and their roles in gene regulation with implica- tions for development in mammals. Below we discuss H3K4 methyltransferases and H3K4 methylation in mouse and humans with their regulatory roles in gene expression. H3K4 methylation and HMTs in mouse and humans Histones are among the most conserved proteins dur- ing evolution of eukaryotes. As discussed for other eukaryotes, roles of histone methylation in gene regu- lation and development are largely conserved in mam- mals. Genomic studies have revealed that both mouse and humans have  30 000 genes, and mouse has orthologs for 99% of human genes (Mouse Genome Sequencing Consortium, 2002). Given the close conser- vation between these two systems, we have reviewed progresses made towards H3K4 methylation and the corresponding HMTs in both mouse and humans. H3K4 trimethylation patterns in mammals are similar to yeast, and are associated with transcriptional start sites. H3K4 dimethylation, however, has a distinct dis- tribution pattern. Genomic mapping studies have revealed that H3K4 dimethylation overlaps with H3K4 trimethylation in the vicinity of active genes [131,132]. However, significant H3K4 methylation is also observed at the inactive genes within b-globin locus. This observation suggests that H3K4 methylation may have important roles in maintaining the transcriptional ‘poised state’ [131], in addition to its role in transcrip- tional stimulation. Although H3K4 methylation is usually localized to punctuate sites, a unique pattern of H3K4 methylation is found at the HOX gene cluster in mammals. Broad regions of continuous H3K4 methylation spanning multiple genes with the intergenic regions are observed at HOX gene clusters in mouse and humans [132]. Unlike in yeast, which has just one H3K4 methyltrans- ferase, mammals have at least 10 (Table 1) [33,133– 138]. Of these, six members containing the SET domain (MLL1, MLL2, MLL3, MLL4, SET1A and SET1B) belong to the mixed lineage leukemia (MLL) family bearing homology to yeast Set1 and Drosophila TRX (Tables 2 and 3) [30,33,49,133,134]. Other H3K4 methyltransferases identified are ASH1, SET7 ⁄ 9, SMYD3 and a meiosis-specific factor, Meisetz [33,133– 138]. The presence of several HMTs in humans might be because of the role of different HMTs at different developmental stages in determining cell fate. The MLL family of HMT proteins has an important role in regulating HOX gene expression. This is particularly significant because deregulation of HOX genes is asso- ciated with leukemia via rearrangements in the MLL1 gene. In addition to HOX genes, MLL1 also targets non-HOX genes like p27 and p18 [139]. Interestingly, deletions or truncations in MLL1, MLL2 and MLL3 have different phenotypes in mice [140–144]. For example, deletion of MLL1 shows misregulation in a number of HOXA genes, including HOXA1 [140–143], whereas MLL2 controls expression of HOXB2 and HOXB5, and loss of MLL3 causes severe growth retar- dation and widespread apoptosis [141–143]. Thus, MLL1, MLL2 and MLL3 appear to have nonredun- dant functions. As is true for yeast and other eukaryotes, MLL fam- ily HMTs are assembled into multi-subunit complexes (Table 2). These complexes have three common subun- its, WD repeat domain 5 (WDR5), retinoblastoma binding protein 5 (RBPB5) and Drosophila ASH2-like (ASH2L) [49,116,117,119,120,133,143] which form the core of the complex. MLL SET is active only when it associates with the core complex, a feature reminiscent of S. cerevisae COMPASS [145]. The WDR5 subunit of the MLL complex is essential for binding of MLL HMT to dimethylated-K4 of histone H3. It is also a key player in the conversion of di- to trimethylation of histone H3 at K4. Consistent with this observation, a reduced level of H3K4 trimethylation is observed following knockdown of WDR5. Consequently, HOX gene expression in human cells is decreased signifi- cantly in the absence of WDR5 [146]. Thus, WDR5 appears to play a crucial role in regulating the actitivi- ties of MLL HMTs. In addition to its role in H3K4 methylation, MLL complex interacts with a TATA-box binding protein (TBP)-associated factor component of transcription factor IID, components of E2F transcription factor 6 (E2F6) subcomplex, and MOF (a MYST family S. Malik and S. R. Bhaumik H3K4 methyltransferases from yeast to human FEBS Journal 277 (2010) 1805–1821 ª 2010 The Authors Journal compilation ª 2010 FEBS 1813 histone acetyl transferase involved in histone H3 K16 acetylation) [147]. The interaction between the MLL1 complex and MOF is particularly interesting as both histone H3 K4 methylation and histone H4 K16 acety- lation are marks of active transcription, and hint towards the coordinated actions of histone H4 K16 acetylation and H3K4 methylation in transcriptional regulation. Indeed, it has been shown that MLL1 HMT and male absent on the first (MOF) HAT acti- vities are required for the proper expression of HoxA9 [145,147]. Further, the MLL3 ⁄ MLL4 complex coor- dinates H3K4 methylation with demethylation of histone H3 at K27 through its UTX subunit [49,133,134,148,149]. Furthermore, H3K4 methylation is coupled to histone deacetylation by the interaction of HMTs SET1 ⁄ ASH2 with SIN3 deacetylase [116]. Thus, HMTs in human appears to play diverse func- tions in regulation of gene expression, and hence development. In addition to its role in mammalian development, H3K4 methylation also participates in the maintenance of pluripotency. It has been demonstrated that embry- onic stem cells maintain ‘bivalent domains’ of repres- sive (histone H3 K27 methylation) and activating (H3K4 methylation) marks. The bivalent domains silence developmental genes in embryonic stem cells while still preserving their ability to be activated in response to appropriate developmental cues [150]. However, bivalent domains are also maintained at other genes in fully differentiated cell types [151,152]. Methylation is dynamically regulated by demethylas- es like LSD1 and JmjC domain-containing enzymes. Demethylase, LSD1, can demethylate mono- and dimethylated-K4 of histone H3 (Table 4). LSD1 has been shown to interact with repressors like (co)repres- sor for element-1-silencing transcription factor and activation complexes like MLL1. These observations implicated that MLL1 is involved in transcriptional activation as well as repression [33,49,153,154]. Deme- thylation of trimethylated-K4 of histone H3 is cata- lyzed by JmjC domain-containing proteins. Mammals have four JARID family members with the JmjC domain (Table 4). These are JARID1A or Rbp2, JARID1B or PLU-1, JARID1C or SMCX and JAR- ID1D or SMCY [33,155]. Both H3K4 methyltransfe- rases and demethylases function in a coordinated fashion to delicately regulate H3K4 methylation, and hence gene expression. The significance of understanding regulation of H3K4 methylation is exemplified by the occurrence of cancers and other diseases following mutations of H3K4 methyltransferases and demethylases or their altered expression [33]. For example, MLL1 is translo- cated in leukemia; SMYD3 is overexpressed in colorec- tal cancer, liver, breast and cervical cancers; and the demethylase, LSD1, is overexpressed in prostate can- cer. The implication of HMTs and demethylases in cancer and human diseases has led to the idea of utiliz- ing them as therapeutic targets. In fact, biguanide and bisguanidine polyamine analogs inhibit the demethy- lase, LSD1, in human colon carcinoma cells. Inhibition of LSD1 facilitates expression of the aberrantly silenced genes in cancer cells [156]. In addition to tar- geting a single enzyme, epigenetic therapy combining related proteins ⁄ pathways is a promising alternative. Thus, combinatorial therapy targeting HMTs, demeth- ylases, histone deacetylases, DNA methyltransferases and others may work efficiently in treating diseases caused by epigenetic misregulations. In fact, a combi- natorial therapy targeting histone deacetylases and DNA methyltransferases has been shown to have a synergistic role in gene regulation, and clinical trials have consistently yielded encouraging results [157–161]. Structural and functional studies have shown specific similarities and differences among the HMTs in diverse organisms. In mammals, the core components of the MLL complex, RBBP5, WDR5 and ASH2L, form a structural platform with which the catalytic SET1 can associate, whereas Set1 in S. cerevisiae is required for the integrity of the COMPASS complex. In S. cerevisi- ae, Cps60 ⁄ Bre2 does not interact directly with Cps50 ⁄ Swd1. However, in mammals their homologs RBBP5 and ASH2L interact strongly in the complex. These different interactions imply diverse regulatory mechanisms for the HMTs. This suggests that in higher eukaryotes, the core complex can be similar, but different subunits can associate with this core com- plex at different stages of cell development to provide HMT activity. Thus, there are several HMT complexes in higher eukaryotes dedicated to diverse cellular roles. Further, the C-terminal SET domain is invariant in different HMTs, although the N-terminal domains are divergent. This enables the HMTs to associate with a broad spectrum of proteins to ensure the downstream events. Given the increasing complexity in higher eukaryotes, the diversity of H3K4 HMTs is not sur- prising. However, the conservation of the fundamental SET domain in these HMTs is intriguing. In addition, many of the H3K4 HMTs share several domains ⁄ com- ponents, indicating a common mechanism of action. 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Malik. protein; EcR, ecdysone receptor; HAT, histone acetyl transferase; H3K4, histone H3 lysine 4; HMT, histone methyltransferase; MLL, mixed lineage leukemia; MOF,