Tài liệu Báo cáo khoa học: Mixed lineage leukemia: roles in gene expression, hormone signaling and mRNA processing docx

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-

mia 1 (MLL1), MLL2, MLL3, MLL4, MLL5, hSet1A,
Keywords
epigenetics; estrogen receptor; gene
expression; histone methyltransferase;
hormone signaling; mixed lineage leukemia;
mRNA processing; NR-box; nuclear
receptor; SET domain
Correspondence
S. S. Mandal, Gene Regulation and Disease
Research Laboratory, Department of
Chemistry and Biochemistry, The University
of Texas at Arlington, Arlington, TX 76019,
USA
Fax: +1 817 272 3808
Tel: +1 817 272 3804
E-mail:
(Received 14 November 2009, revised 16
January 2010, accepted 28 January 2010)
doi:10.1111/j.1742-4658.2010.07606.x
Mixed lineage leukemias (MLLs) are an evolutionarily conserved trithorax
family of human genes that play critical roles in HOX gene regulation and
embryonic development. MLL1 is well known to be rearranged in myeloid
and lymphoid leukemias in children and adults. There are several MLL
family proteins such as MLL1, MLL2, MLL3, MLL4, MLL5, Set1A and
Set1B, and each possesses histone H3 lysine 4 (H3K4)-specific methyltrans-
ferase activity and has critical roles in gene activation and epigenetics.
Although MLLs are recognized as major regulators of gene activation,
their mechanism of action, target genes and the distinct functions of differ-
ent MLLs remain elusive. Recent studies demonstrate that besides H3K4
methylation and HOX gene regulation, MLLs have much wider roles in

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.

Because of their importance in gene regulation and dis-
ease, MLLs have been isolated from human cells and
their protein–protein interaction profiles and enzymatic
activities have been characterized in detail [5,11]. These
studies demonstrate that MLL1, MLL2, MLL3,
MLL4, Set1A and Set1B exist as distinct multiprotein
complexes with several common subunits including
Ash2, Wdr5, Rbbp5 and Dpy30. Each of these MLLs
and Set1 contains a catalytic SET domain responsible
for their HMT activity (Fig. 2). Recently, we demon-
strated that human CpG-binding protein (CGBP)
interacts with MLL1, MLL2 and human Set1, and is a
core component of these HMT complexes [12]. In
addition to the core components, MLLs interact with
various unique components including chromatin-
remodeling factors, mRNA-processing factors and
nuclear hormone receptors. Dou et al. [5] purified an
H3
H4
H2A
H2B
K9
K4
Me
Me
Me
Me
Me
K79
K36

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

Silencing
H2A
K119
Ub
Nucleosome
Fig. 1. Mixed lineage leukemias (MLL) are
histone H3 at lysine 4-specific methylases
that regulate gene activation.
MLL3
MLL5
MLL1
MLL4
MLL2
CXXC zf
FYRC FYRN
BROMO
HMG
AT-Hook
PHD
NR-box (LXXLL)
Taspase1
RING
SET
Fig. 2. Domain structures of mixed lineage leukemias (MLLs). AT-hook is a DNA-binding domain. Bromodomains (BROMO) are involved in
the recognition of acetylated lysine residues in histone tails. CXXC-zf is a Zn-finger domain involved in protein–protein interactions. FYRC and
FYRN domains are involved in heterodimerization between MLL
N
and MLL
C
terminal fragments. High-mobility group (HMG) domains are

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

the H3K4-specific HMT activity of the MLL1 complex
in vitro. Removal of Wdr5 completely abolishes the
methylation activity of MLL complex. In contrast to
Wdr5, Rbbp5 and Ash2, results from our laboratory
demonstrate that knockdown of CGBP abolishes the
recruitment of MLL1 into the promoter of its target
HOXA7 gene, affecting H3K4 trimethylation and
HOXA7 gene expression [12]. These observations sug-
gest that MLL-interacting components have distinct
roles in controlling MLL-mediated histone methylation
and target gene expression.
MLLs are critical for HOX gene
regulation and embryonic development
Homeobox genes are a group of evolutionarily con-
served genes that encode transcription factors and reg-
ulate gene expression during development. There are
more than 200 homeobox genes in vertebrates that are
classified into two major groups, class I and II. Class I
homeobox-containing genes share a high degree of
identity and are known as HOX genes. Human
encodes 39 different HOX genes that are clustered in
four different groups, HOXA–D, located on chromo-
somes 7, 17, 12 and 2, respectively. Based on sequence
similarities and location within the cluster, HOX genes
are further classified into 13 paralogous groups [17].
The nature of the body structures depends on the spe-
cific combination of HOX gene products and the
expression of specific HOX gene varies at different
stages of development. Therefore, proper regulation
and maintenance of HOX genes are essential for nor-

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

defects and alteration in the maintenance of the proper
transcription levels of several target Hox loci (such
Hoxd4, a5 and a7) during development [22]. Impor-
tantly, these changes in gene expression levels are
associated with a reduction of histone H3K4 monome-
thylation (H3K4me1) and altered DNA methylation
patterns at the same Hox loci. These results demon-
strate an essential role for the MLL-SET domain in
chromatin structure and Hox gene regulation in vivo
[22]. Using an inducible knockout system, Jude et al.
[23] investigated the roles of Mll1 in adult hematopoi-
etic stem cells (HSCs) and progenitors. These studies
demonstrated that Mll1 is essential for the maintenance
of adult HSCs and progenitors, with fetal bone marrow
failure occurring within 3 weeks of Mll1 deletion. HSCs
lacking Mll1 exhibit ectopic cell-cycle entry resulting in
the depletion of quiescent HSCs, and Mll1 deletion
in myelo-erythroid progenitors results in reduced proli-
feration and a reduced response to cytokine-induced
cell-cycle entry [23]. Committed lymphoid and myeloid
cells no longer require Mll1, indicating Mll1-dependent
early multipotent stages of hematopoiesis [23]. These
Biochemical functions of MLL K. I. Ansari and S. S. Mandal
1792 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
studies demonstrate that Mll1 plays selective and inde-
pendent roles within the hematopoietic system, main-
taining quiescence in HSCs and promoting proliferation
in progenitors [23]. Similarly, in an independent study
using a conditional knockout mouse model, it was
shown that Mll1, although dispensable for the produc-

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

the later stages of differentiation [30]. These observa-
tions suggest that the associations of either MLL or
MLL-associated coregulators with the promoters are
modulated at different stages of development, resulting
in differential expression of target HOX genes. Overall,
various knockout and cell line studies demonstrate that
MLLs are master players in HOX gene regulation and
development.
MLLs are general transcriptional
regulator (beyond HOX genes)
Although MLLs are well-recognized as master regula-
tors of HOX genes, studies suggest that MLLs play
much wider roles in regulating the transcription of
diverse gene types [31–34]. Several approaches have
been used to investigate MLL target genes. However,
it is important to note that the functions of MLLs
may be highly dependent upon the cellular environ-
ment (such as the presence of hormones and nutrients),
cell types and developmental stages. Analyzing the
functions of MLLs in gene regulation in a given cell
lineage is important and may provide crucial informa-
tion about the roles of MLLs in that particular cellular
environment. However, this may underscore much
wider functions of MLLs in other cell types or under
different cellular environments and therefore the regu-
latory roles of MLLs may not be generalized based on
information obtained from experiments with a single
cell type.
Using a genome-wide promoter binding assay it has
been shown that MLL1 and H3K4 trimethylation is

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

+ ⁄ +
) and Mll1
) ⁄ )
mouse embryonic fibro-
blasts (MEFs). These studies demonstrated that Mll1
is required for the H3K4 trimethylation of < 5% of
promoters carrying this modification [37]. Although
Mll1 is only required for the methylation of a subset
of Hox genes, menin, a component of the Mll1 and
Mll2 complexes, is required for the overwhelming
majority of H3K4 methylation at Hox loci [37]. How-
ever, the loss of Mll3 ⁄ Mll4 and ⁄ or the Set1 complexes
has little to no effect on the H3K4 methylation of Hox
loci or on their expression levels in these MEFs [37].
These observations suggest that different MLLs may
have distinct functions beyond H3K4 methylation.
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 1793
Another surprising but interesting recent observation
may significantly alter the view of transcriptional regu-
lation [38]. Using genome-wide analyses in embryonic
stem cells (also differentiated cells), Guenther et al.
[38] showed that the promoter-proximal nucleosome of
most of the protein-coding genes is trimethylated at
H3K4, although it is generally thought to be the mark
of only transcriptionally active genes. Furthermore,
most of the genes considered to be inactive (because of
low transcript levels) experience transcription initiation
and associated histone modification [38]. These obser-
vations suggest that transcription initiation is a general

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

, dissociates from condensed mitotic
chromatins, migrates from the nucleus to the cyto-
plasm and returns at the end of telophase when the
nucleus starts to relax. However, the global level of
MLL1 is not affected [40]. We also found that several
MLL target HOX genes (such as HOXA10, HOXA5
and HOXB7) are expressed differentially during cell-
cycle progression. For example, HOXA10 expression is
very high in the S phase, decreases significantly in
G
2
⁄ M and is completely absent in G
1
[40]. Expression
of HOXA5 increases from very low levels at the begin-
ning of the S phase, reaches a maxima at G
2
⁄ M,
declining sharply to its initial low level and remaining
so throughout mitosis and G
1
. Importantly, MLL1
binds to the promoter of these HOX genes as a func-
tion of their expression during cell-cycle progression
[40]. These observations suggest that although at the
microscopic level, MLLs dissociate from the con-
densed nuclear matrix during mitosis, some MLL
remains associated with chromatin and maintains
expression of specific cell-cycle-related genes during
mitosis. Depletion of MLL1 also results in cell-cycle

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

deoxynivalenol and chromatin immunoprecipitation
analysis demonstrated increased binding of MLL1 to
the target HOX gene promoters in the presence of
deoxynivalenol [41]. These observations indicate the
possible involvement of MLLs in the stress response.
The relationship between MLL and stress is further
strengthened by observations that several external
stresses (such as exposure to estrogen or flavonoids)
induce the rearrangement of MLL1 [42,43]. Similarly,
exposure to contraceptive pills increases the risk of leu-
kemia in the fetus and infants [44]. These observations
suggest that MLLs and associated diseases are linked
with different types of stress.
MLLs are also found to be associated with the telo-
meres. MLLs affect H3K4 methylation and transcrip-
tion of telomere in a length-dependent manner [31].
RNAi-mediated depletion of MLL in human diploid
fibroblasts affects telomere chromatin modification,
telomere transcription, telomere capping and induces
the telomere damage response. Overall, these studies
Biochemical functions of MLL K. I. Ansari and S. S. Mandal
1794 FEBS Journal 277 (2010) 1790–1804 ª 2010 The Authors Journal compilation ª 2010 FEBS
demonstrated that MLLs are not only critical for
HOX gene regulation, but also are associated with
other types of gene regulation.
Are MLLs merely histone methylases or
do they have additional roles in gene
regulation?
Gene expression may have different states: basal, acti-
vated (usually stimulated by external stimuli such as

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

that the MLL1 core complex uses a nonprocessive
mechanism to catalyze multiple lysine methylation [46].
Nevertheless, methylation of histones by MLLs plays
key roles in the transactivation of target genes. In
general, MLL1 interacts and colocalizes with RNAPII
primarily at the promoter during transcription. In
some cases, MLL1 is also found to be associated
within the coding region of a subset of actively tran-
scribed target genes and loss of MLL1 function
impairs RNAPII distribution [30]. These observations
indicate that an intimate association of MLL and
RNAPII is required for transcription initiation and ⁄ or
the elongation of MLL target genes [30,47].
In addition to their direct roles in gene activation
via H3K4 methylation, MLLs interact with other chro-
matin modifying enzymes and coregulators (see below)
and facilitate gene expression. For example, MLL1
complex physically interacts with acetyl transferase
MOF which remodels chromatin by histone acetylation
and charge neutralization [15]. Both H3K4 methylation
and H4K16 acetyl transferase activities are required
for optimal transactivation of the MLL1 target
HOXA9 gene [15]. The MLL1 C-terminal domain is
also an interaction partner for histone acetyl transfer-
ase CREB-binding protein (CBP) and the INI1 subunit
of SWI ⁄ SNF chromatin remodeling complexes, sug-
gesting further coordination of MLL complexes in his-
tone methylation, acetylation, chromatin remodeling
and mRNA synthesis [48,49].
MLL1 fusion proteins are also associated with vari-

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

involved in MLL-mediated transactivation of the target
gene. For example, the AT-hook DNA-binding
domains present in MLLs indicate that they mediate
targeting of MLLs to their nuclear site and permit spe-
cific binding to the minor groove of AT-rich DNA [56].
Deletion of AT-hook motifs substantially impairs the
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 1795
transforming effects of MLL–ENL on primary myeloid
progenitors [57,58]. In addition to the AT-hook, the
CXXC finger domain present in the N-terminus of
MLL mediates selective binding of MLL to nonmethy-
led-CpG DNA [59]. The CXXC domain recruits MLL–
ENL to nonmethylated CpG DNA sites in vitro and
affects transactivation of target genes in vivo [59]. Our
recent study showed that CGBP containing CXXC
DNA-binding motifs interacts with MLLs and recruits
them into the promoter of the HOXA7 gene [12]. DNA
methyltransferase homology regions present in MLL1
may have an affinity for AT- and GC-rich sequences
and play critical roles in the recruitment of MLL to
target genes. In addition to AT-hooks and the CpG-
binding activity of MLL and its interacting protein
CGBP, recruitment of MLLs to a target gene promoter
may be influenced by various other interacting proteins
such as Wdr5 and Menin [5,13,30]. Wdr5 recognizes
the histone H3K4 methyl-mark introduced by MLL1
and it has therefore been suggested that Wdr5 ensures
the processivity of MLL1-mediated histone modifica-
tion [60]. Similar to Wdr5, Menin binds to the N-termi-

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-

tains one highly variable transactivation region (AF1)
and the C-terminal region contains a conserved trans-
activation domain AF2, which undergoes structural
changes in response to hormones and ultimately results
in activation of the NR. Activated NRs bind to the
promoters of target genes leading to their activation.
It is well-recognized that during ligand-dependent
transcription activation, activated NRs require various
types of coregulators (coactivators and corepressors)
[64,65]. For example, during transcriptional activation
of E2-responsive genes, estrogen receptors (ER) associ-
ate with a distinct subset of cofactors, depending on
the target gene, binding affinities and relative abun-
dance of these factors in the cells [65]. These coactiva-
tors and repressors usually exist in multiple complexes,
possess multiple enzymatic activities and (in a simpli-
fied view) bridge ERs, to chromatin components such
as histone, to components of the basal transcription
machinery or to both [66]. Intense research has identi-
fied a large number of cofactors including three mem-
bers of the SRC-1 family (SRC-1, SRC-2 ⁄ GRIP1 ⁄ TIF2
and SRC-3 ⁄ AIB1 ⁄ ACTR ⁄ pCID ⁄ RAC3 ⁄ TRAM1),
CBP, p ⁄ CAF, thyroid hormone receptor protein and
vitamin D3 receptors-interacting proteins. Studies have
demonstrated that ASCOM, which consists of MLLs
as an interacting component, also participates actively
in E2-mediated gene activation [67,68]. In addition,
Menin, which is also a component of MLL1 ⁄ MLL2
complexes, acts as a coregulator for ERa and regulates
estrogen-responsive genes [13].

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

ERa, recruits MLL2 complex into the promoter of
estrogen-responsive genes (TFF1 gene) and regulates
their expression in an estrogen-dependent manner.
Menin serves as a critical link between activated ERa
and the MLL2–coactivator complex in this process.
A similar interaction involving Menin was observed in
the case of peroxisome proliferator-activated receptor
(PPARgamma, which generally expresses in several
MEN1-related tumor cells) and regulates their target
gene expression in a ligand-dependent manner [71].
In addition to Menin, other components of the
MLL complex or MLL-interacting complexes may
recruit MLLs onto the gene promoter in a ligand-
dependent manner. Lee et al. showed that ASCOM
complexes containing MLL3 or MLL4 are tightly colo-
calized in the nucleus [67,72]. Their study also revealed
that the C-terminal SET domain of MLL3 and MLL4
directly interacts with INI1, an integral subunit of AT-
Pase-dependent chromatin remodeling complex
SWI ⁄ SNF [67] and their mutational studies revealed
that both ASCOM and SWI ⁄ SNF complex facilitate
each other binding to the promoter of NR target gene.
Thus, these studies suggest that in addition to direct
interactions of MLLs with NRs, they interact via vari-
ous MLL-interacting components in a ligand-depen-
dent manner to regulate NR-mediated gene expression.
MLLs interact with different NRs via ASC2
complexes and regulate NR target gene
activation
A widely studied NR coactivator is activating signal

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

Menin
+1
Wdr5Wdr5
CGBPCGBP
Rbbp5
Dpy30Dpy30
NRNR
L
MLL
ASC2/
INI1
Ash2Ash2
Wdr5Wdr5
MeninMenin
CGBPCGBP
Rbbp5
Dpy30Dpy30
MLL
Ash2Ash2
LXXLL
domain of
MLL
Menin
L
ASC2ASC2
Chromatin modification/remodeling
Gene activation
NR-
coregulators
??

??
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

promoter via interaction with RAR. Along with
ASC2, other ASCOM components including MLLs
(MLL3 and MLL4) are recruited to the RAR-2 pro-
moter and lead to H3K4 trimethylation, chromatin
remodeling and gene activation in a retinoic acid-
dependent manner. The presence of an intact LXXLL
domain is essential for ligand-dependent recruitment of
ASC2 and other ASCOM components to the RAR-2
promoter suggesting direct ligand-dependent interac-
tion between ASC2 NR box (likely NR box 1) and
RAR [6,75]. The NR box 1 of ASC2 also shows rela-
tively weak yet specific interaction with the farne-
soid X receptor during transactivation of FXR [32]. By
contrast, in the case of LXR, the NR box 2 of ASC2
specifically recognizes LXRs [6] and recruits MLL3
and MLL4 to the LXR target gene, sterol regulatory
element binding protein 1c (SREBP-1c). Mutation of
ASC2 ablated the effect of LXR ligand (T1317) on
LXR target gene expression by affecting H3K4 trime-
thylation. However, mutation of MLL3 partially
suppressed expression of the target genes [6].
Lee et al. [75] demonstrated that independent knock-
down of MLL3 and MLL4 results in attenuation of
retinoic acid-induced H3K4 trimethylation, but does
not abolish it completely. However, parallel knock-
down of both MLL3 and MLL4 suppresses retinoic
acid-induced expression of RAR-b2 [75]. These obser-
vations suggest that MLL3 and MLL4 are present in
independent ASCOM complexes and are redundant in
histone methylation [75]. This redundancy in their

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

acids affect Hox gene expression markedly and pro-
duce homeotic transformation [77]. Retinoic acid regu-
lates expression of 3¢ Hox paralogs including Hoxa1
and Hoxb1 during development of the central nervous
system in early embryogenesis. The well-defined
boundaries of different sections of the brain are devel-
oped by endocrine regulation of developmental gene
expression, including selected Hox genes [78].
Although, retinoids regulate anterior Hox genes, recent
data showed that posterior Hox genes are regulated by
estrogens and progesterone [79]. Neonatal exposure to
diethylstilbestrol downregulated uterine Hoxa10
expression [80]. Hoxb13, which is associated with the
normal differentiation and secretary function of the
mouse ventral prostate, is suppressed upon exposure to
neonatal estrogen [81]. Ovariectomy in mouse affects
the expression of Hoxc6, which is critical for mam-
mary gland development and milk production [82].
Expression of Hoxa10 in canine glandular epithelium,
embryo, luminal epithelium and uterus fluctuates over
different stages of pregnancy, whereas Hoxa10 is
significantly upregulated by either estrogen or proges-
terone. Similar to retinoic acid, estrogens regulate
expression of the 5¢ Hox paralogs such as Hoxa9,
Hoxa10 and Hoxa11, which are expressed in posterior
and distal domains of the body axis [83]. Although the
hormonal regulation of several HOX genes is driven
by developmental processes and MLLs are well known
as key regulators of HOX genes, the roles of MLLs in
Biochemical functions of MLL K. I. Ansari and S. S. Mandal

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

recruiting MLLs into the HOXC13 promoter [84].
Although further investigations are needed to under-
stand the detailed mechanism of MLL-mediated
HOXC13 and other HOX genes regulation, these
studies demonstrate that MLL histone methylases, in
coordination with nuclear hormone receptors, do play
critical roles in the regulation of steroid hormone-
mediated HOX genes and this mechanism of hormonal
regulation of HOX genes may be linked with HOX
gene regulation during development and diseases.
Do MLLs have any roles in mRNA
processing?
In eukaryotes, gene expression involves various steps
such as transcription (synthesis of RNA), mRNA
processing (such as mRNA capping, splicing, poly-
adenylation and cleavage), surveillance and export of
the matured mRNA from the nucleus to the cytoplasm
for translation [1]. Diverse studies involving genetic
and mutational analysis demonstrate that transcription
is tightly coupled with mRNA processing [87].
RNAPII is the key player in coordinating these co-
transcriptional events via orchestrated recruitment of
transcription and mRNA-processing factors through-
out transcription. Although an emerging view is that
all the steps from transcription, mRNA processing and
translation are mechanically and functionally coupled,
the proteins involved in this coupled process are still
poorly characterized [87].
MLLs are primarily recognized as having critical
roles in gene activation via H3K4 methylation of pro-

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,

estrogen) not only change the expression of their target
genes by binding and modulating the activity of their
nuclear receptors (NRs), but also modulate alternative
splicing events for different genes [90]. For example, ste-
roid hormone estrogen (E2) modulates the expression of
splice variants of the genes encoding ERa, VGEF and
Oxytocin [90]. Given the complexity of steroid hormone
signaling, multiple modes of action may operate in
making alternate splicing decisions. These include post-
translational modification of splicing factors and pro-
moter-dependent recruitment of splicing factors via
transcriptional coregulators. In fact, NR coregulators
are shown to interact with spliceosome and couple tran-
scription with alternative splicing [90]. Several protein
candidates have been linked with alternative splicing
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 1799
decisions. For example, ERa interacts with SF3a, p120
and other components of snRNPs, and controls
E2-dependent alternate splicing of the E2-responsive
gene, oxytocin [90]. NRs also interact with the SR
(serine- and arginine-rich proteins) family of splicing
factors. CAPER, a SR-like protein, interacts with ERa
and the progesterone receptor and modulates the
ligand-dependent transcription and alternate splicing of
target genes [91]. Depletion of CAPER (by siRNA)
altered the effect of progesterone on alternative splicing
of endogenous vascular endothelial growth factor
transcripts indicating its critical role in the process.
Similar to snRNPs, the hnRNP family of splicing fac-

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

regulating gene activation beyond their HMT activi-
ties. MLLs are master players in both basal and acti-
vated transcription, especially under an hormonal
environment. Although MLLs are found to interact
with various mRNA-processing factors directly or
EFs
SF
SF
SF
GpppN
Me
CTD
GpppN
Me
Coregulators
GTF
RNAPII
Me
Me
Me Me
MLL
SF
SF
CE
ASC2
RNAPII
Me
Me
S5-P
S2-P

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-

plored. Because steroid hormones and nuclear recep-
tors are intimately associated with cell differentiation,
embryonic development and diverse types of human
disease, including cancer and cardiovascular diseases,
MLLs are likely linked with all those key physiological
events and human diseases beyond their well-recog-
nized roles in HOX gene regulation and mixed lineage
leukemia.
Acknowledgements
We thank Imran Hussain, Sahba Kasiri, Bishakha
Shrestha, Saoni Mandal and other Mandal lab mem-
bers for useful discussion and critical comments. This
work was supported in parts by ARP (00365-0009-
2006) and American Heart Association (0765160Y). I
apologize for not being able to cite many references of
my colleagues due to a citation limit.
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