MINIREVIEW
Mixed lineage leukemia: roles in gene expression, hormone
signaling and mRNA processing
Khairul I. Ansari and Subhrangsu S. Mandal
Department of Chemistry and Biochemistry, The University of Texas at Arlington, TX, USA
Introduction
In eukaryotes, gene regulation is a complex process [1].
In addition to RNA polymerase II (RNAPII), there are
numerous other transcription factors and regulatory
proteins that coordinate with RNAPII to accurately
express a particular gene under a specific cellular envi-
ronment. In higher organisms, DNA is complexed with
various histones and other nuclear proteins in the form
of compact chromatins. These chromatins are not easily
accessible to gene expression machinery unless they are
modified or remodeled [1]. Intense research over the
past two decades has led to the discovery of various
chromatin-remodeling factors and histone-modifying
enzymes that modulate chromatin structures to facilitate
gene expression [1]. Histone methyltransferases (HMTs)
are key enzymes that introduce methyl groups into the
lysine side chain of histone proteins and regulate gene
activation and silencing (Fig. 1). Histone H3 lysine 4
(H3K4) methylation is an evolutionarily conserved
mark with fundamental roles in gene activation [2]. Set1
is the only H3K4-specific HMT present in yeast and is a
component of a multiprotein complex called COM-
PASS [3]. In higher eukaryotes, H3K4-specific HMTs
are diverged with increased structural and functional
complexity [4]. In humans, there are at least eight
H3K4-specific HMTs that include mixed lineage leuke-

gene activation and regulate diverse other genes. Interestingly, several
MLLs interact with nuclear receptors and have critical roles in steroid-
hormone-mediated gene activation and signaling. In this minireview, we
summarize recent advances in understanding the roles of MLLs in gene
regulation and hormone signaling and highlight their potential roles in
mRNA processing.
Abbreviations
ASCOM, activating signal cointegrator-2 (ASC2) complex; CBP, CREB-binding domain; CGBP, CpG-binding protein; ER, estrogen receptor;
H3K4, histone H3 lysine 4; HMT, histone methyltransferase; HSC, hematopoietic stem cell; LXR, liver X receptor; MLL, mixed lineage
leukemia; NR, nuclear receptor; RAR, retinoic acid receptor; RNAPII, RNA polymerase II; SF, splicing factor.
1790 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
hSet1B and ASH1 [5]. The high conservation and multi-
plicity of MLLs suggests that they have crucial and
distinct functions in the cell, although their detailed
mechanisms of action are largely unknown. Recent stud-
ies have demonstrated that MLLs are key epigenetic reg-
ulators of diverse gene types associated with cell-cycle
regulation, embryogenesis and development. MLLs also
interact with nuclear receptors (NR) and coordinate
hormone-dependent gene regulation, suggesting their
crucial roles in reproduction, organogenesis and disease
[6]. In this minireview, we summarize recent advance-
ments addressing the roles of MLLs in human gene reg-
ulation, hormone signaling and mRNA processing.
MLLs are human H3K4-specific
methyltransferases
It is well-known that MLLs are often rearranged,
amplified or deleted in different types of cancer [7–10].
MLLs are master regulators of HOX genes, which are
key players in embryogenesis and development.

K27
K20
K120
Me
K119
H3
H4
H2A
H2B
K9
K4
H3
MLL
MeMe
MeMe
MeMe
MeMe
MeMe
K79
K36
K27
Activation
Activation
Silencing
K20
K120
Ub
H2B
MeMe
H4

involved in binding DNA with low sequence specificity. LXXLL domains (also known as the NR box) are involved in interaction with nuclear
receptor (NR). Plant homodomain (PHD) and RING fingers are usually involved in protein–protein interactions. The SET domain is responsible
for histone lysine methylation. The Taspase 1 site is the proteolytic site for the protease Taspase 1. Some other domains including frequent
coiled coil domains that mediate homo-oligomerization are not shown.
K. I. Ansari and S. S. Mandal Biochemical functions of MLL
FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS 1791
MLL1 complex that contains histone acetyl transfer-
ase, MOF, host cell factors, HCF1 and HCF2. Simi-
larly, Menin, which is a product of the MEN1 tumor
suppressor gene, is an interacting component of MLL1
and MLL2 complexes [13].
MLL-associated HMT activity appears to be func-
tional only in the context of their multiprotein com-
plexes and each MLL-interacting protein plays a
distinct role in regulating MLL-mediated histone
methylation and gene activation. For example, Wdr5
binds to dimethylated H3K4 and knockdown of Wdr5
results in decreased expression of MLL1 target HOX
genes without affecting binding of MLL1 complexes to
the promoters [14]. Similarly, knockdown of Rbbp5 or
Ash2 also reduces the expression of HOX genes with-
out affecting the recruitment of MLL1 into their pro-
moter [5,15]. Independent studies have demonstrated
that Wdr5, Rbbp5 and Ash2 are essential for HOX
gene expression and this requirement lies in the regula-
tion of H3K4 dimethylation and trimethylation [16].
In vitro reconstitution experiments have demonstrated
that Wdr5, Rbbp5, Ash2 and the catalytic C-terminus
of MLL1 (MLL-C) form a functional MLL1 HMT
complex [5]. The absence of Rbbp5 and Ash2 reduces

mal physiological functions and growth.
To understand the developmental function of
MLL1 and its role in HOX gene regulation, several
groups have used different strategies to disrupt the
MLL1 gene. These studies have shown that homo-
zygous Mll1 (a murine ortholog of human MLL1)
knockout mice die during embryogenesis [18,19].
Lethality at embryonic day 10.5 is associated with
multiple patterning defects in neural crest-derived
structures of the branchial arches, cranial nerves and
ganglia [18,19]. MLL protein is critical for proper
regulation of HOX genes during development.
Notably, expression of several examined Hox genes
is correctly initiated in Mll1-null (Mll1
) ⁄ )
) mice, but
is not sustained as the function of Mll1 becomes
necessary, leading to embryonic lethality [18,19].
Mll1-mutant mice also exhibit hematopoietic abnor-
malities, associated with decreased expression of a
number of Hox genes (Hoxa7, Hoxa9, Hoxa10,
Hoxa4) in the Mll1-mutant fetal liver [20,21]. The
early embryonic lethality of Mll1 homozygous
mutants has prevented detailed analysis of the role
of MLL1 function during adult development and
hematopoiesis.
Interestingly, deletion of the SET domain (responsi-
ble for HMT activity) alone of Mll1 is not lethal and
mutant mice are fertile. Homozygous SET domain-
truncated mutants exhibit developmental skeletal

tion of mature adult hematopoietic lineages, plays a
critical role in stem cell self-renewal in fetal liver and
adult bone marrow [24].
The critical role of MLL1 in HOX gene regulation is
evident from a simple fibroblast model and HOX gene
expression is dependent on the HMT activities of
MLL [25,26]. Myeloid transformation by MLL oncog-
enes is associated with expression of a specific subset
of HOXA genes [27]. Murine primary myeloid progeni-
tor cell lines immortalized by five different MLL fusion
proteins exhibit a characteristic Hoxa gene expression
profile and all lines expressed Hoxa7, Hoxa9, Hoxa10
and Hoxa11 genes located at the 5¢-end of the Hoxa
cluster [28]. By contrast, 3¢-end Hoxa genes were vari-
ably expressed with periodicity, as evidenced by low
levels of Hoxa1 , higher levels of Hoxa3 and Hoxa5
and the complete absence of Hoxa2, Hoxa4 and
Hoxa6 expression [28]. These studies also demon-
strated that Hoxa7 and Hoxa9 are required for
efficient in vitro myeloid immortalization by an MLL
fusion protein, but not other leukemogenic fusion
proteins [28]. In an independent study, depletion of
Taspase1 (a MLL1-specific protease that cleaves pre-
MLL1 peptide to generate functional MLL1 protein
fragments) diminished expression of selected HOX
genes across the HOXA cluster [29]. Despite continu-
ous expression of MLL1 throughout hematopoiesis,
MLL target genes HOXA7, HOXA9 and MEIS1 are
expressed during early hematopoietic lineages and their
expression downregulated to undetectable levels during

enriched at the promoters of transcriptionally active
genes [35]. The overlap of MLL1 binding and H3K4
trimethylation reinforces the role of MLL1 as a posi-
tive global regulator of gene transcription [35]. MLL1
also localizes to microRNA (miRNA) loci that are
involved in leukemia and hematopoiesis [35]. MLL
associates only with transcriptionally active promoters
and therefore is cell-type and differentiation-stage spe-
cific [30]. In a separate study, using gene expression
profiling in murine cell lines (Mll
+ ⁄ +
and Mll
) ⁄ )
), it
was shown that Mll1 is associated with both transcrip-
tionally active and repressed genes [36]. These studies
also demonstrated that beyond HOX genes, Mll1 regu-
lates diverse other gene types that are involved in dif-
ferentiation and organogenesis pathways (such as
COL6A3, DCoH, gremlin, GDID4, GATA-6 and
LIMK) [36]. p27kip1 and GAS-1, which are known
tumor suppressor proteins involved in cell-cycle regula-
tion, are also found as targets of Mll1 [36]. Mll1 is also
linked to the expression of a variety of genes linked
with leukemogenesis and other malignant transforma-
tions including HNF-3 ⁄ BF-1, Mlf1, FBJ, Tenascin C,
PE31 ⁄ TALLA-1 and tumor protein D52-like gene [36].
More recently, Wang et al. [37] performed a genome-
wide analysis of H3K4 methylation patterns in wild-
type (Mll1

phenomenon in most genes and the elongation phase
perhaps contributes significantly to the regulation of
transcript synthesis.
In addition to the genome-wide studies, independent
studies from several laboratories demonstrate that
MLLs play critical roles in cell-cycle regulation. Take-
da et al. [34] showed that mutation of Taspase 1 results
in the downregulation of cyclin E, A and B, and upreg-
ulation of p16 (an S-phase inhibitor). MLL1 binds to
the promoters of cyclin E1 and E2 and there is a
marked reduction in the H3K4 trimethylation level, as
well as MLL1 occupancy at the cyclin E1 and E2 pro-
moters in Taspase 1-negative cells [34]. MLLs also
interact with other cell-cycle regulatory transcription
factors such as E2F family proteins. Whereas MLL1
interacts with E2F2, E2F4 and E2F6, MLL2 interacts
with E2F2, E2F3, E2F5 and E2F6 [34]. Similar to
E2Fs, the G
1
-phase regulator HCF-1 recruits MLL1
and Set1 to E2F-responsive promoters and induces his-
tone methylation and transcriptional activation during
the G
1
phase [39]. Recently, we demonstrated that
MLL1 and H3K4 trimethylation have distinct dynam-
ics during cell-cycle progression [40]. MLL1, which is
normally associated with transcriptionally active
chromatins in G
1

arrest at the G
2
⁄ M phase and inhibits cellular
growth, further suggesting its crucial roles in cell-
cycle progression [40]. Although the detailed roles of
MLLs and their interacting proteins in cell-cycle
regulation are still not clear, multiple lines of
evidence indicate that MLLs play critical roles in
cell-cycle regulation.
In addition to cell-cycle regulatory genes, MLL1
plays important roles in the regulation of stress-
responsive genes. MLL3 and MLL4 act as a p53 coac-
tivator (a tumor suppressor gene) and are required for
H3K4 trimethylation and expression of endogenous
p53 target genes in response to the DNA-damaging
agent, doxorubicin [32]. Expression of p21, a promi-
nent p53 target gene, was significantly reduced in
MLL3-depleted mice relative to wild-type mice.
Although direct interaction of MLLs with p53 leads to
transcription activation in vitro [15], Menin mediates
recruitment of MLLs onto the promoter of p27 and
p18 genes affecting their expression [33]. Depletion of
MLL1 leads to p53-dependent growth arrest [31].
Recent studies from our laboratory demonstrate that
MLLs are upregulated upon exposure to oxidative
stress induced by a common food contaminant myco-
toxin, deoxynivalenol [41]. Transcription factor Sp1
plays a critical role in deoxynivalenol-mediated upreg-
ulation of MLL1. MLL-targeted HOX genes (such as
HOXA7) are also upregulated upon exposure to

temperature, special nutrients, hormones or other
stresses) and repressed transcription (silencing) [1].
Although the requirement of general transcription fac-
tors is likely to be similar in both the basal and acti-
vated transcription environment, the requirement for
accessory factors (activators and coactivators, repres-
sors and corepressor) may vary depending on the tran-
scription state and cell type. We hypothesize that
although all MLLs are H3K4-specific HMTs, they
have distinct functions during basal and activated tran-
scription. H3K4 methylation is likely to be a common
requirement for both basal and activated transcription.
However, in addition to or even independent of their
H3K4-specific HMT activity, MLLs may have differ-
ent coregulatory functions during activated (and possi-
bly repressed) transcription. Based on some of our
recent unpublished observations, MLL1 appears to act
as a histone methylase during basal transcription
whereas MLL2, MLL3 and MLL4 replace MLL1
during activated transcription and act as coactivators,
at least in selected HOX genes.
MLLs contain diverse functional domains (Fig. 2).
Studies indicate that the SET domain plays pivotal
roles in transcriptional regulation in target genes. Dele-
tion of the MLL1 SET domain abolishes its ability to
activate HOX gene expression, indicating key roles for
H3K4 methylation by the SET domain during transac-
tivation [45]. Kinetic studies revealed that the reaction
leading to H3K4 dimethylation involves the transient
accumulation of a monomethylated species, suggesting

ous chromatin remodeling factors and transcriptional
regulators. For example, MLL1 is fused to ace-
tyl transferase CBP and related protein P300, espe-
cially in therapy-induced secondary leukemia.
Structure–function analysis demonstrated that bromo
and acetyl transferase domains are necessary and suffi-
cient for the oncogenic transformation of respective
proteins [50,51]. The MLL fusion protein MLL–AF10
interacts with SWI ⁄ SNF complex via GAS41 and INI1
[52]. The MLL fusion protein MLL–ENL also associ-
ates and cooperates with SWI ⁄ SNF complexes to acti-
vate transcription of HOX genes [53]. Furthermore,
ENL (MLL fusion partner) is associated not only with
MLL fusion protein AF4 family members (AF4,
AF5q31, LAF4) but also with positive transcription
elongation factor-b and histone H3K79-specific meth-
yltransferase DOT1L [54,55]. Interestingly, the MLL
fusion partner AF10 binds to DOT1L and that
DOT1L recruitment was necessary for the oncogenic
transforming activity of MLL–AF10 [55]. H3K79
methylation was dramatically increased in the HOXA9
gene upon activation by MLL–ENL. In summary,
these studies suggest that coordination of histone mod-
ification (including methylation and acetylation) and
nucleosome remodeling by MLL complexes in both
wild-type and MLL fusion proteins results in balanced
transactivation of target genes.
In addition to the catalytic SET domain, several
other protein–DNA or protein–protein interacting
domains present in MLL peptides are functionally

nus of MLL1 and facilitates recruitment of MLL1, and
several oncogenic MLL1 fusion proteins, to target gene
promoters [61]. Menin can be recruited to DNA via
interactions with sequence-specific transcription factors
such as NRs (discussed below) and with the chromatin-
associated factor lens epithelium-derived growth factor,
a chromatin-associated protein required for both MLL-
dependent transcription and leukemic transformation.
Thus, diverse DNA-binding domains, protein–protein
interaction modes and pre-existing chromatin modifica-
tions may facilitate the binding of MLLs, depending on
the context and cell types, to facilitate transcription
activation. The detailed functions of other MLL1
domains are summarized Cosgrove and Patel in this
minireview series [62].
MLLs are key players in nuclear
receptor-mediated gene activation and
hormone signaling
NRs are a special class of transcription factors that
are responsible for sensing the presence of hormones
in cells and transducing signals for various cellular
pathways, including the activation of hormone-respon-
sive genes in a hormone-dependent manner [63]. Most
NRs share a common structural organization that
includes a DNA-binding domain, a ligand-binding
domain and a transactivation domain. The DNA-bind-
ing domain is responsible for DNA binding specificity
and dimerization, and the ligand-binding domain is
responsible for binding of the ligand and associated
induced functions. The N-terminal region of NRs con-

MLLs interact with NR via NR boxes and regulate
gene activation
NR coactivators characteristically contain helical
LXXLL or FXXLF motifs (NR box) and interact with
the AF2 domain of the liganded NR [63,64,69].
Sequence analysis demonstrates that MLL histone
methylases (MLL1–4) contain one or more NR boxes
(Fig. 2). MLL1 contains one NR box, whereas MLL2,
-3 and -4 contain three to four, indicating their potential
interaction with NRs and associated gene regulation.
Recent studies demonstrate that MLLs act as coacti-
vators for various hormone-responsive genes in a
ligand-dependent manner. Mo et al. [70] demonstrated
that MLL2 interacts physically with estrogen receptor-
alpha (ERa), a critical player in estrogen-mediated gene
activation, via its LXXLL motifs in the presence of the
steroid hormone estrogen. Disruption of the interaction
between ERa and MLL2 (using MLL2 siRNA) inhibits
estrogen-mediated transactivation of estrogen-respon-
sive genes such as cathepsin D and pS2. MLL2 is
Biochemical functions of MLL K. I. Ansari and S. S. Mandal
1796 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
recruited to the promoters of cathepsin D and pS2
along with ERa in an E2-dependent manner.
In addition to the direct interaction of MLL2 with
ERa, MLLs may interact with ERs via other MLL-
interacting proteins (NR-box containing) such as
Menin, ASC2 and INI1, and regulate target gene acti-
vation (Fig. 3). Dreijerink et al. [13] showed that
Menin (through its LXXLL domains) interacts with

cointegrator-2 (ASC2, also named AIB3, TRBP,
TRAP250, NRC, NCOA6 and PRIP) [69]. ASC2 is a
coactivator of multiple nuclear receptors including reti-
noic acid receptor (RAR), liver X receptors (LXR)
+1
Wdr5
CGBP
Rbbp5
Dpy30
NRNR
L
MLL
ASC2/
INI1
Ash2
Wdr5
Menin
CGBP
Rbbp5
Dpy30
Ash2
LXXLL
domain of
MLL
Menin
L
ASC2
NR-
coregulators
ASC2/INI1

??
ASC2/INI1
Menin
HRE
Fig. 3. Mixed lineage leukemias (MLLs) are coregulators for nuclear receptor (NR)-mediated gene activation. During hormone-mediated gene
activation, NRs bind to the hormone and are activated. The activated NRs, along with various coregulators, bind to the hormone response
elements present in the promoters of hormone-responsive genes leading to their gene activation. Usually proteins containing an LXXLL
domain interact with NRs and act as coregulators for NR-mediated gene activation. MLLs (MLL1–4) contain one or more LXXLL domains.
Therefore, MLLs may interact directly with NRs via their own LXXLL domain(s) and regulate NR-mediated gene activation. Alternatively,
MLLs might interact with NRs via different MLL-interacting proteins such as ASC2, Menin, INI1 that contain multiple LXXLL domains. In
addition to NR, there are various other NR coregulators (other than MLL and ASCOM) that, in coordination with NRs, have essential roles in
NR-mediated gene activation. However, it is not yet clear if MLLs interact and ⁄ or coordinate with any of these NR coregulators (CBP ⁄ P300,
PCAF, SRC-1 family, etc.) in a ligand-dependent manner to regulate NR-target genes.
K. I. Ansari and S. S. Mandal Biochemical functions of MLL
FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS 1797
and ER [6]. ASC2 contains two LXXLL domains
through which it interacts with NRs in a ligand-depen-
dent manner. ASC2 is present in a steady-state multi-
protein complex, ASCOM, that also contains various
other proteins including MLL histone methylases and
MLL-interacting proteins Rbbp5, Wdr5 [6,73]. More
recent studies identified additional ASCOM-specific
components that include PTIP, PTIP-associated pro-
tein-1 and UTX (a H3K27-specific demethylase) [74].
Thus, ASCOM contains two distinct groups of histone
modifier that are linked to transcriptional activation
[73,75] and ASC2 bridges nuclear receptors and these
histone modifiers [6,32,76].
During retinoic acid-induced activation of RAR-2
(a RAR target gene), ASC2 is recruited to the RAR-2

function was further confirmed by depleting the com-
mon subunits of ASCOM3 (containing MLL3) and
ASCOM4 (containing MLL4) complexes [75]. The
siRNA-mediated depletion of Wdr5, Rbbp5 and
Ash2L caused significant suppression of RAR-medi-
ated H3K4 trimethylation of the RAR-b2 gene.
Because both MLL3 and MLL4 in ASCOM complexes
are recruited by NR box 1 of ASC2, depletion of
ASC2 results in impaired recruitment of both MLL3
and MLL4 affecting H3K4 trimethylation and RAR
target gene expression [75]. These results suggest that
the key function of ASC2 in transactivation is to pres-
ent MLL3 and MLL4 to the target gene promoter. In
addition to acting as an anchor between NR and
ASCOM complexes, ASC2 also confers specificity on
different ASCOM complexes towards different hor-
mone-induced genes [75]. For example, depletion of
Menin, a common component of MLL1 and MLL2,
does not affect expression of RAR-2 in mouse embry-
onic fibroblasts [75]. However, depletion of ASC2
leads to not only impaired expression of RAR-2, but
also suppression of H3K4 trimethylation, indicating
that RAR-2 is a specific target for MLL3 and MLL4
but not for MLL1 and MLL2.
Are MLLs involved in hormonal regulation of
HOX genes?
Expression of the HOX genes is tightly regulated
throughout development. Studies suggest that
hormones play critical roles in the regulation of devel-
opmental genes, including HOX. For example, retinoic

1798 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
hormonal regulation of HOX genes are largely
unknown.
In an effort to understand the mechanism of HOX
gene regulation, especially under hormonal environ-
ments, we found that several HOX genes including
HOXC10 and HOXC13 are transcriptionally regulated
by estrogen [84]. Hoxc13 is a critical gene involved in
the regulation of hair keratin gene cluster and alope-
cia, and Hoxc13-mutant mice lack external hair, sug-
gesting a critical role in hair development [85].
Although, steroid hormones are critical players in sex-
ual differentiation and both HOXC13 and steroid hor-
mones are linked with hair follicle growth and
difference in hair patterning in males and females, the
roles of steroid hormones in HOXC13 gene regulation
are unknown [86]. Our studies demonstrate that
HOXC13 is transcriptionally activated by estrogen
(E2). HOXC13 promoter contains several estrogen
response elements and ERs bind to these in an
E2-dependent manner [84]. Knockdown of estrogen
receptors, ERa and ERb, suppresses E2-mediated acti-
vation of HOXC13. Similarly, knockdown of histone
methylase MLL3 suppressed E2-induced activation of
HOXC13 [84]. MLL histone methylases (MLL1–4)
were bound to the promoter of HOXC13 in an
E2-dependent manner [84]. Furthermore, knockdown
of either ERa or ERb affected the E2-dependent bind-
ing of MLLs (MLL1-4) into HOXC13 estrogen
response elements, suggesting critical roles for ERs in

moters. H3K4 trimethyl mark is thought to recruit
various transcriptional coregulators during transcrip-
tion activation. Trimethylation of histone H3 at
lysine 4 localizes primarily at the 5¢ region of genes
and is tightly associated with active loci. Recently,
Reinberg and colleagues demonstrated that H3K4-
trimethylated polypeptide specifically binds CHD1, a
chromatin remodeling factor involved in transcription
elongation [88]. Using a conventional biochemical
purification approach, they demonstrated that CHD1
exists as a stable complex with components of the
spliceosome. Knockdown of CHD1 by siRNA reduced
the association of U2 snRNP components with chro-
matin, affecting the efficiency of pre-mRNA splicing
on active genes in vivo [88]. Studies in yeast, Drosophila
and mammalian systems also demonstrate a role for
CHD1 in transcript elongation and termination. These
studies suggest that methylated H3K4 serves to facili-
tate the competency of pre-mRNA maturation through
bridging spliceosomal components to H3K4-trimethyl
via CHD1. In addition, MLL complexes are also
shown to coordinate Ski-complex that are critical play-
ers in mRNA splicing suggesting further link between
MLLs with transcription and mRNA processing [89].
In most eukaryotic genes, exons are separated by
introns, and introns need to be spliced out prior to
translation. Splicing is carried by a spliceosome that
consists of 100–300 different proteins. Increasing
amounts of evidence suggest that transcriptional stimuli,
such as steroid hormones (i.e. androgens, progestins,

tors also interacts with NRs to modulate the transcript
synthesis and maturation [90].
Although many possibilities exist in the decision-
making process of E2-mediated alternative splicing of
ER-target genes, we hypothesize that MLL histone
methylases may be linked with alternative splicing
(Fig. 4). In particular, ASC2 (a component of the AS-
COM complex that also contains MLLs), which
confers target gene specificity to MLL complexes,
interacts with CoAA (a hnRNP-like protein) and
CAPER that are key players in alternate splicing [92].
Because diverse studies demonstrate that MLL histone
methylases (especially MLL2, MLL3 and MLL4) inter-
act with NRs via critical involvement of ASCOM com-
plexes that interact with factors involved in alternative
splicing, it is likely that MLLs also coordinate the
process of alternative splicing especially in hormone-
regulated genes.
Conclusion
MLL histone methylases are critical players in gene
regulation and disease. The high conservation and
multiplicity of MLLs in higher eukaryotes indicate that
they have crucial and distinct roles in gene activation
and other cellular events. Although the discovery of
MLLs and characterization of their HMT activities
and protein–protein interaction profiles have shed sig-
nificant light on their mechanism of action in gene
activation, their detail functions in the regulation of
different types of genes are yet to be revealed. In
general, MLLs appear to have much wider roles in

GpppN
Me
CTD
Me
Me
Me
Pre-initiation phase
Elongation phase
Fig. 4. Recruitment of splicing factors (SFs) with the pre-initiation complex at the promoter via interaction with mixed lineage leukemias
(MLLs) or other coregulators. Because several SFs are found to interact with MLL or ASCOM complexes (containing MLLs and ASC2), it is
likely that some of these SFs are recruited to the promoter (via interaction with MLLs or other coregulator complexes or with RNAPII) prior
to the transcription initiation forming pretranscription initiation complexes (containing RNAPII, general transcription factors, mRNA capping
enzymes, regulators and coregulators). Once the RNAPII moves to the transcription elongation phase, these SFs move onto the splice sites
of the nascent pre-mRNA to execute splicing in a co-transcriptional manner. S5-P and S2-P denote the phosphorylation states of the RNAPII
C-terminal domain at the transcription imitation and elongation phases, respectively. As some of the coregulators are specific to activated
transcription (especially in presence of hormones or other stimuli), splicing and alternative splicing decisions (hence recruitment of SFs into
the pre-initiation complexes) may be linked with hormone signaling. Red and green methyl groups represent H3K4 trimethylation and H3K36
dimethylation during transcription initiation and elongation phases respectively. EFs represent elongation factors.
Biochemical functions of MLL K. I. Ansari and S. S. Mandal
1800 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
indirectly, and we hypothesize that MLLs are potential
key players in mRNA processing, their functional
details in mRNA-processing events remain to be eluci-
dated. The presence of the LXXLL domain in different
MLLs makes them attractive candidates for interaction
with nuclear hormone receptors and associated gene
regulation. Even though studies demonstrate that
MLLs are critical players in diverse types of NR-medi-
ated gene regulation, their critical roles and interplay
with different NR coregulators remain largely unex-

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