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

M I N I R E V I E W

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

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: smandal@uta.edu

(Received 14 November 2009, revised 16 January 2010, accepted 28 January 2010)

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.

doi:10.1111/j.1742-4658.2010.07606.x

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,

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.

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MLL

MeMe Me

H3 H3

K9 K9

H3

K4 K4

K79 K36 K27 K79 K36 K27

H4 H4

K20 K20

H4

Silencing

Activation

H2A H2A

Silencing

Activation

K120 K120

H2B H2B

K119 K119

Ub

H2A

H2B

Ub

Nucleosome

Fig. 1. Mixed lineage leukemias (MLL) are histone H3 at lysine 4-speciﬁc methylases that regulate gene activation.

in embryogenesis

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

components

including

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 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 that MLL1, MLL2, MLL3, studies demonstrate 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 these HMT complexes [12]. In core component of addition to the core components, MLLs interact with various unique chromatin- remodeling factors, mRNA-processing factors and nuclear hormone receptors. Dou et al. [5] puriﬁed an

MLL1

MLL2

MLL3

MLL4

MLL5

AT-Hook

FYRC

FYRN

CXXC zf

HMG

BROMO

Taspase1

NR-box (LXXLL)

RING

PHD

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 MLLN and MLLC 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.

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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].

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

role

during

leading to embryonic

in the Mll1-mutant

fetal of Mll1

embryonic

lethality

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 for proper ganglia [18,19]. MLL protein is critical of HOX genes regulation 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, lethality [18,19]. Mll1-mutant mice also exhibit hematopoietic abnor- malities, associated with decreased expression of a (Hoxa7, Hoxa9, Hoxa10, number of Hox genes Hoxa4) liver [20,21]. The early homozygous mutants has prevented detailed analysis of the role of MLL1 function during adult development and hematopoiesis.

exhibit developmental

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 in regulating MLL-mediated histone distinct 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

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 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

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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 it was using a conditional knockout mouse model, 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].

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.

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,

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- (such as ferentiation and organogenesis pathways 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 is required for the overwhelming Mll2 complexes, 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.

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interacting proteins

clear, multiple

still not

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 cellular the G2 ⁄ M phase and inhibits arrest at growth, further suggesting its crucial roles in cell- cycle progression [40]. Although the detailed roles of in cell-cycle MLLs and their regulation are lines of evidence indicate that MLLs play critical roles in cell-cycle regulation.

roles

important

in the regulation of

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.

from several

associated with transcriptionally

that

In addition to cell-cycle regulatory genes, MLL1 plays 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 to wild-type mice. relative 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 [31]. MLL1 leads to p53-dependent growth arrest Recent studies from our laboratory demonstrate that MLLs are upregulated upon exposure to oxidative stress induced by a common food contaminant myco- [41]. Transcription factor Sp1 toxin, deoxynivalenol 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 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.

In addition to the genome-wide studies, independent laboratories demonstrate that studies 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 G1-phase regulator HCF-1 recruits MLL1 and Set1 to E2F-responsive promoters and induces his- tone methylation and transcriptional activation during the G1 phase [39]. Recently, we demonstrated that MLL1 and H3K4 trimethylation have distinct dynam- ics during cell-cycle progression [40]. MLL1, which is normally active chromatins in G1, 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 G2 ⁄ M and is completely absent in G1 [40]. Expression of HOXA5 increases from very low levels at the begin- the S phase, reaches a maxima at G2 ⁄ M, ning of declining sharply to its initial low level and remaining so throughout mitosis and G1. 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

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

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for demonstrated that MLLs are not only critical 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?

transactivation of

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 transferase complex physically interacts with acetyl MOF which remodels chromatin by histone acetylation and charge neutralization [15]. Both H3K4 methylation and H4K16 acetyl transferase activities are required the MLL1 target for optimal 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].

secondary

therapy-induced

Gene expression may have different states: basal, acti- vated (usually stimulated by external stimuli such as special nutrients, hormones or other temperature, 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- type. We hypothesize that scription state and cell 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.

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 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.

example,

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 the catalytic SET domain, several other protein–DNA or protein–protein interacting in MLL peptides are functionally domains present involved in MLL-mediated transactivation of the target gene. For 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

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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

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

NR coactivators contain helical characteristically LXXLL or FXXLF motifs (NR box) and interact with the liganded NR [63,64,69]. the AF2 domain of 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.

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

Recent studies demonstrate that MLLs act as coacti- vators in a for various hormone-responsive genes 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

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recruited to the promoters of cathepsin D and pS2 along with ERa in an E2-dependent manner.

(NR-box containing)

chromatin

remodeling

[13]

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- complex Pase-dependent 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

In addition to the direct interaction of MLL2 with ERa, MLLs may interact with ERs via other MLL- such as interacting proteins Menin, ASC2 and INI1, and regulate target gene acti- vation (Fig. 3). Dreijerink et al. 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

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- liver X receptors (LXR) noic acid receptor (RAR),

Dpy30 Dpy30 Dpy30

Rbbp5 Rbbp5

NR- NR- coregulators coregulators

Dpy30 Dpy30 Dpy30

Ash2Ash2 Ash2

CGBP CGBP CGBP

ASC2ASC2 ASC2

Wdr5Wdr5 Wdr5

MLL MLL

Wdr5 Wdr5Wdr5

CGBP CGBP CGBP

MLL

Ash2 Ash2Ash2

ASC2/ ASC2/ INI1 INI1

Menin MeninMenin

ASC2/INI1 ASC2/INI1

Menin Menin

Rbbp5 Rbbp5

Menin Menin

LXXLL LXXLL domain of domain of MLL MLL

L L

+1 +1

NRNR NRNR

HRE

Chromatin modification/remodeling

Gene activation

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.

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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].

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?

Studies

suggest

development.

genes

including

selected Hox

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].

the HOX genes is tightly regulated Expression of that throughout 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, [78]. Although, retinoids regulate anterior Hox genes, recent data showed that posterior Hox genes are regulated by estrogens and progesterone [79]. Neonatal exposure to uterine Hoxa10 diethylstilbestrol downregulated 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 stages of pregnancy, whereas Hoxa10 is different 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

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- the RAR-b2 gene. ated H3K4 trimethylation of Because both MLL3 and MLL4 in ASCOM complexes are recruited by NR box 1 of ASC2, depletion of

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regulation of HOX genes are

largely

hormonal unknown.

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].

role

in hair development

linked with hair

studies demonstrate

(MLL1-4)

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].

regulation,

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 [85]. Although, steroid hormones are critical players in sex- ual differentiation and both HOXC13 and steroid hor- follicle growth and mones are difference in hair patterning in males and females, the roles of steroid hormones in HOXC13 gene regulation are unknown [86]. Our 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- into HOXC13 estrogen ing of MLLs 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 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 tightly coupled with mRNA processing [87]. is RNAPII is the key player in coordinating these co- transcriptional events via orchestrated recruitment of

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

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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

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].

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

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

Pre-initiation phase

Elongation phase

S2-P

S5-P

ASC2

F S

CTD

E

M L L

Fs

Coregulators

Me

GTF

CE

RNAPII

F S

Me Me Me GpppN GpppN GpppN

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.

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7 Marschalek R (2010) Mixed lineage leukemia: roles in human malignancies and potential therapy. FEBS J doi: 10.1111/j.1742-4658-2010.07608.x.

8 Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J, Ben Abdelali R, Macintyre E, De Braekeleer E, De Braekeleer M et al. (2009) New insights to the MLL recombinome of acute leukemias. Leukemia 23, 1490– 1499.

9 Thomas M, Gessner A, Vornlocher HP, Hadwiger P, Greil J & Heidenreich O (2005) Targeting MLL–AF4 with short interfering RNAs inhibits clonogenicity and engraftment of t(4;11)-positive human leukemic cells. Blood 106, 3559–3566. 10 Horton SJ & Williams O (2006) The mechanism of

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.

hematopoietic progenitor cell immortalization by MLL– ENL. Cell Cycle 5, 360–362.

Acknowledgements

11 Hess JL (2004) MLL: a histone methyltransferase disrupted in leukemia. Trends Mol Med 10, 500– 507. 12 Ansari KI, Mishra BP & Mandal SS (2008) Human

CpG binding protein interacts with MLL1, MLL2 and hSet1 and regulates Hox gene expression. Biochim Biophys Acta 1779, 66–73. 13 Dreijerink KM, Mulder KW, Winkler GS, Hoppener

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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