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.. Abbreviat
Trang 1Mixed 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.
Trang 2DNA-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
79
me A
B
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
H4
N-S G R G K G G K G L G K G G A K R H R K V -C
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.
Trang 3The 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 rangmaintain-ing
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 transcriptioC-termi-nal 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)
ASH1 ASH2 TRR
MLL1 MLL2 MLL3 MLL4 SET1A SET1B SET7 ⁄ 9 SMYD3 ASH1 Meisetz
Trang 4Cps35⁄ 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].
Trang 5homologs 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 pombeshows 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 me2⁄ 1, di- and monomethyl.
Saccharomyces cerevisae Schizosaccharomyces pombe Caenorhabditis elegans Drosophila Mammals (mouse and human)
Swm2 (me 2 )
SPR-5 (me 2 ) T08D10.2 (me 2 ) R13G10.2 (me2)
Lid (me 3 ) LSD1 (me 2 ⁄ 1 )
JARID1A (me 3 ⁄ 2 ) JARID1B (me3⁄ 2) JARID1C (me3⁄ 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
TRR
Menin; HCF1 ⁄ HCF2; NCOA6; PA1;
PTIP; UTX; MOF; SET7 ⁄ 9; SMYD3; Meisetz
Trang 6H3K4 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 elegansis 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-ganswas 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 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 F5SWD-2B11.1⁄ 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
Trang 7more 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
Drosophilahas long been a model organism for studying
developmental processes, because development in
humans and Drosophila are homologous processes
Dro-sophilaand 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 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
Trang 8histone 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,
Trang 9AT-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
Drosophilais 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
Trang 10histone 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 enzymdemethylas-es
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 deacetdemeth-ylases, 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
Concluding remarks
Studies in several eukaryotes demonstrate the conser-vation of H3K4 HMTs from yeast to humans The roles of H3K4 methylation in gene regulation,