Mixed lineage leukemia: roles in gene expression, hormone signaling and mRNA processing

Authors


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

Abstract

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 methyltransferase 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 different 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

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 environment. 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 COMPASS [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 leukemia 1 (MLL1), MLL2, MLL3, MLL4, MLL5, hSet1A, hSet1B and ASH1 [5]. The high conservation and multiplicity of MLLs suggests that they have crucial and distinct functions in the cell, although their detailed mechanisms of action are largely unknown. Recent studies have demonstrated that MLLs are key epigenetic regulators 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 advancements addressing the roles of MLLs in human gene regulation, hormone signaling and mRNA processing.

Figure 1.

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

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 disease, 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 demonstrated 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 MLL1 complex that contains histone acetyl transferase, MOF, host cell factors, HCF1 and HCF2. Similarly, Menin, which is a product of the MEN1 tumor suppressor gene, is an interacting component of MLL1 and MLL2 complexes [13].

Figure 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.

MLL-associated HMT activity appears to be functional only in the context of their multiprotein complexes 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 without affecting the recruitment of MLL1 into their promoter [5,15]. Independent studies have demonstrated that Wdr5, Rbbp5 and Ash2 are essential for HOX gene expression and this requirement lies in the regulation 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 suggest 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 conserved genes that encode transcription factors and regulate 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 chromosomes 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 specific 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 normal 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 homozygous 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 abnormalities, 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 (responsible 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]. Importantly, these changes in gene expression levels are associated with a reduction of histone H3K4 monomethylation (H3K4me1) and altered DNA methylation patterns at the same Hox loci. These results demonstrate 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 hematopoietic 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 proliferation 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 studies demonstrate that Mll1 plays selective and independent roles within the hematopoietic system, maintaining 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 production 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 oncogenes is associated with expression of a specific subset of HOXA genes [27]. Murine primary myeloid progenitor 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 variably 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 demonstrated 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 continuous 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 observations 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 regulators 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 environment (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 information 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 regulatory 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 positive 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 specific [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 transcriptionally active and repressed genes [36]. These studies also demonstrated that beyond HOX genes, Mll1 regulates diverse other gene types that are involved in differentiation 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 regulation, 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 transformations 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 fibroblasts (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]. However, 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.

Another surprising but interesting recent observation may significantly alter the view of transcriptional regulation [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 observations 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. Takeda et al. [34] showed that mutation of Taspase 1 results in the downregulation of cyclin E, A and B, and upregulation 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 promoters 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 histone methylation and transcriptional activation during the G1 phase [39]. Recently, we demonstrated that MLL1 and H3K4 trimethylation have distinct dynamics during cell-cycle progression [40]. MLL1, which is normally associated with transcriptionally active chromatins in G1, dissociates from condensed mitotic chromatins, migrates from the nucleus to the cytoplasm 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 beginning of the S phase, reaches a maxima at G2/M, 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 function of their expression during cell-cycle progression [40]. These observations suggest that although at the microscopic level, MLLs dissociate from the condensed 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 G2/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 coactivator (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 prominent 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 mycotoxin, deoxynivalenol [41]. Transcription factor Sp1 plays a critical role in deoxynivalenol-mediated upregulation 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 leukemia 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 telomeres. MLLs affect H3K4 methylation and transcription 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 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, activated (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 factors is likely to be similar in both the basal and activated transcription environment, the requirement for accessory factors (activators and coactivators, repressors and corepressor) may vary depending on the transcription state and cell type. We hypothesize that although all MLLs are H3K4-specific HMTs, they have distinct functions during basal and activated transcription. 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 different coregulatory functions during activated (and possibly 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. Deletion of the MLL1 SET domain abolishes its ability to activate HOX gene expression, indicating key roles for H3K4 methylation by the SET domain during transactivation [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 transcribed 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 chromatin 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 transferase CREB-binding protein (CBP) and the INI1 subunit of SWI/SNF chromatin remodeling complexes, suggesting further coordination of MLL complexes in histone methylation, acetylation, chromatin remodeling and mRNA synthesis [48,49].

MLL1 fusion proteins are also associated with various chromatin remodeling factors and transcriptional regulators. For example, MLL1 is fused to acetyl transferase CBP and related protein P300, especially in therapy-induced secondary leukemia. Structure–function analysis demonstrated that bromo and acetyl transferase domains are necessary and sufficient 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 associates and cooperates with SWI/SNF complexes to activate 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 methyltransferase 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 modification (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 specific binding to the minor groove of AT-rich DNA [56]. Deletion of AT-hook motifs substantially impairs the 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 nonmethyled-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 modification [60]. Similar to Wdr5, Menin binds to the N-terminus 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 modifications 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-responsive 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-binding 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 contains one highly variable transactivation region (AF1) and the C-terminal region contains a conserved transactivation 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) associate with a distinct subset of cofactors, depending on the target gene, binding affinities and relative abundance of these factors in the cells [65]. These coactivators and repressors usually exist in multiple complexes, possess multiple enzymatic activities and (in a simplified view) bridge ERs, to chromatin components such as histone, to components of the basal transcription machinery or to both [66]. Intense research has identified a large number of cofactors including three members 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 ERα 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 coactivators for various hormone-responsive genes in a ligand-dependent manner. Mo et al. [70] demonstrated that MLL2 interacts physically with estrogen receptor-alpha (ERα), 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 ERα and MLL2 (using MLL2 siRNA) inhibits estrogen-mediated transactivation of estrogen-responsive genes such as cathepsin D and pS2. MLL2 is recruited to the promoters of cathepsin D and pS2 along with ERα in an E2-dependent manner.

In addition to the direct interaction of MLL2 with ERα, MLLs may interact with ERs via other MLL-interacting proteins (NR-box containing) such as Menin, ASC2 and INI1, and regulate target gene activation (Fig. 3). Dreijerink et al. [13] showed that Menin (through its LXXLL domains) interacts with ERα, 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 ERα 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].

Figure 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.

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 colocalized 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 ATPase-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 various MLL-interacting components in a ligand-dependent 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 retinoic acid receptor (RAR), liver X receptors (LXR) and ER [6]. ASC2 contains two LXXLL domains through which it interacts with NRs in a ligand-dependent manner. ASC2 is present in a steady-state multiprotein 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 protein-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 promoter 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 interaction between ASC2 NR box (likely NR box 1) and RAR [6,75]. The NR box 1 of ASC2 also shows relatively weak yet specific interaction with the farnesoid 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 trimethylation. However, mutation of MLL3 partially suppressed expression of the target genes [6].

Lee et al. [75] demonstrated that independent knockdown of MLL3 and MLL4 results in attenuation of retinoic acid-induced H3K4 trimethylation, but does not abolish it completely. However, parallel knockdown of both MLL3 and MLL4 suppresses retinoic acid-induced expression of RAR-β2 [75]. These observations 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 common 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-mediated H3K4 trimethylation of the RAR-β2 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 present 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 hormone-induced genes [75]. For example, depletion of Menin, a common component of MLL1 and MLL2, does not affect expression of RAR-2 in mouse embryonic 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 developmental genes, including HOX. For example, retinoic acids affect Hox gene expression markedly and produce homeotic transformation [77]. Retinoic acid regulates 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 developed 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 mammary 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 progesterone. 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 hormonal regulation of HOX genes are largely unknown.

In an effort to understand the mechanism of HOX gene regulation, especially under hormonal environments, 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 alopecia, and Hoxc13-mutant mice lack external hair, suggesting a critical role in hair development [85]. Although, steroid hormones are critical players in sexual differentiation and both HOXC13 and steroid hormones 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, ERα and ERβ, suppresses E2-mediated activation 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 ERα or ERβ affected the E2-dependent binding 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 understand 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, polyadenylation 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 throughout 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 promoters. H3K4 trimethyl mark is thought to recruit various transcriptional coregulators during transcription 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 chromatin, 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 facilitate 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 players 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, steroid hormone estrogen (E2) modulates the expression of splice variants of the genes encoding ERα, 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 promoter-dependent recruitment of splicing factors via transcriptional coregulators. In fact, NR coregulators are shown to interact with spliceosome and couple transcription with alternative splicing [90]. Several protein candidates have been linked with alternative splicing decisions. For example, ERα 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 ERα 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 factors 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 ASCOM 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) interact with NRs via critical involvement of ASCOM complexes 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.

Figure 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.

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 significant 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 activities. MLLs are master players in both basal and activated transcription, especially under an hormonal environment. Although MLLs are found to interact with various mRNA-processing factors directly or indirectly, and we hypothesize that MLLs are potential key players in mRNA processing, their functional details in mRNA-processing events remain to be elucidated. 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-mediated gene regulation, their critical roles and interplay with different NR coregulators remain largely unexplored. Because steroid hormones and nuclear receptors 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-recognized roles in HOX gene regulation and mixed lineage leukemia.

Acknowledgements

We thank Imran Hussain, Sahba Kasiri, Bishakha Shrestha, Saoni Mandal and other Mandal lab members 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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