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Transcriptional activation by MLL fusion proteins in leukemogenesis
Accepted Manuscript Transcriptional activation by MLL fusion proteins in leukemogenesis Akihiko Yokoyama PII:
S0301-472X(16)30697-X
DOI:
10.1016/j.exphem.2016.10.014
Reference:
EXPHEM 3484
To appear in:
Experimental Hematology
Received Date: 12 August 2016 Revised Date:
14 October 2016
Accepted Date: 29 October 2016
Please cite this article as: Yokoyama A, Transcriptional activation by MLL fusion proteins in leukemogenesis, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.10.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Review Article
Department of Hematology and Oncology, Graduate School of Medicine, 54
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Akihiko Yokoyama1, 2
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Transcriptional activation by MLL fusion proteins in leukemogenesis
Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Division of Hematological Malignancy, National Cancer Center Research Institute,
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Tokyo 104-0045, Japan
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Address correspondence to: Akihiko Yokoyama Ph.D.
[email protected] Tel: 81-75-751-3096 Fax: 81-75-751-3201
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Category for Table of Contents: Malignant Hematopoiesis
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Word count: 3,099
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Abstract
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Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause aggressive leukemia. Fusion proteins of MLL and a component of the AF4 family/ENL family/P-TEFb complex (AEP) are responsible for two-thirds of MLL-associated
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leukemia cases. MLL-AEP fusion proteins trigger aberrant self-renewal of
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hematopoietic progenitors by constitutively activating self-renewal-related genes. MLL-AEP fusion proteins activate transcription initiation by loading the TATA-binding protein (TBP) to the TATA element via selectivity factor 1. Although AEP retains
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transcription elongation and mediator recruiting activities, the rate-limiting step activated by MLL-AEP fusion proteins appears to be the TBP loading step unlike
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prevailing views, in which the recruitment of transcription elongation activities are
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emphasized. Here, I review recent advances towards elucidating the mechanisms underlying gene activation by MLL-AEP fusion proteins in leukemogenesis.
Keywords: MLL, Leukemia, AF4, Transcription, Mediator
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Introduction Chromosomal translocations generate fusion genes of the mixed lineage leukemia
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(MLL) gene (also known as MLL1, HRX, HTRX, KMT2A, and ALL1) [1-3] and various partner genes, causing acute leukemia [4-6]. Leukemia involving MLL gene
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rearrangements (hereafter referred to as MLL-r leukemia) accounts for 5–10% of all
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acute leukemia cases and is often accompanied by an unfavorable prognosis [7, 8]. To date, more than 70 MLL fusion genes have been reported, among which MLL fusions with a component of the AF4 family/ENL family/P-TEFb complex (AEP) constitute
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two-thirds of all MLL-r leukemia cases [9, 10]. Because chromosomal translocations of MLL occur within the introns of MLL and its partner gene X, two types of fusion genes
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are generated: MLL-X and X-MLL. In t(4:11) leukemia cases (fusion of MLL and AF4),
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the AF4-MLL fusion gene is often (not always) expressed [11] and its product exhibits oncogenic properties in mouse models [12]. Nevertheless, the MLL-X fusion gene is transcribed in all MLL-r leukemia cases and its product is thought to be the major contributor for oncogenesis [11]. Hereafter, the MLL-X fusion gene will be referred to as the MLL fusion gene. Compared with other cancers, MLL-r leukemia involves fewer genetic alterations [13]. Many mouse disease models have demonstrated that the
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engineered expression of the MLL fusion gene causes leukemia in a relatively short
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period of time [14-16], supporting the notion that the MLL fusion gene is the major driver of leukemogenesis.
MLL fusion proteins cause constitutive expression of a subset of genes
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including HOXA9 and MEIS1, which are normally expressed specifically in immature
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hematopoietic progenitors such as hematopoietic stem cells (HSCs) [17] (Figure 1 A and B). The expression of HOXA9 facilitates the expansion of immature hematopoietic progenitors [18]. Constitutive expression of HOXA9 causes immortalization of
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immature hematopoietic progenitors ex vivo [19], whereas knockout of HOXA9 compromises the capacity to reconstitute the hematopoietic system [20, 21]. HOXA9 is
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highly expressed in most MLL-r leukemia cells [17, 22] and is required for their
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survival [23]. MEIS1, which is also highly expressed in MLL-r leukemia cells, is a cofactor of HOXA9 whose expression levels correlate with leukemia stem cell potential in vivo [24]. Collaborative functions of HOXA9 and MEIS1 appear to be the major driving force of leukemogenesis as co-expression of both genes efficiently induces leukemia in mouse models [18]. Several other MLL target genes including EVI1 and
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PLZF have also been shown to facilitate oncogenic self-renewal of MLL leukemia cells
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[25-27]. MLL fusion proteins also activate the expression of CDK6 [28, 29], the activity of which is required for the exit of HSCs from quiescence [30]. Taken together, these findings demonstrate that MLL fusion proteins confer an HSC-like self-renewing
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property to hematopoietic progenitors via the constitutive activation of various
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self-renewal-related genes, thereby causing leukemia [18, 19, 31]. Therefore, the focus of this article is the mechanism of constitutive gene activation by a group of MLL-AEP
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fusion proteins.
MLL is a transcriptional maintenance factor of cellular memory genes
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MLL is an ortholog of the Drosophila trithorax protein [1, 2]. Both have an
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evolutionarily conserved SET domain with histone methyl transferase (HMT) activity towards histone H3 lysine 4 [32, 33] (Figure 2A). Knockout of MLL results in impaired posterior homeobox (HOX) gene expression [34, 35], similar to the consequences of the genetic ablation of trithorax in Drosophila [36, 37]. HOX genes are referred to as “cellular memory” genes as their position-specific expression is maintained throughout
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development [38, 39]. MLL is not required for the initial activation of HOX genes, but
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is necessary for their continuous expression during development [40]. Accordingly, MLL is considered a transcriptional maintenance factor of cellular memory. MLL is retained on chromatin during mitosis, such that it can efficiently activate its target genes
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in the next G1 phase [41], thereby maintaining the expression of cellular memory genes
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for multiple cell divisions.
MLL activates the expression of posterior HOXA genes in the hematopoietic cell lineage [35, 42]. Posterior HOXA gene expression is highest at the immature
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progenitor stage (e.g., HSCs and multi-potent progenitors [MPPs]) [43], but progressively declines as cells differentiate, and eventually diminishes at the terminally
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differentiated stages (Figure 1B) [44, 45]. MLL promotes proper expansion of the
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immature hematopoietic compartments of both adult and fetal hematopoietic systems presumably by upregulating posterior HOXA gene expression [35, 42, 46, 47]. The effects of MLL deficiency are prominent in situations where hematopoietic progenitors expand rapidly, such as the reconstitution of hematopoietic system [42, 47]. These results indicate that MLL regulates the proper expansion of immature hematopoietic
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progenitors by maintaining the expression of self-renewal-related genes at immature
Mechanisms of target recognition by MLL proteins
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progenitor stages.
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MLL fusion proteins and wild-type MLL regulate a common set of genes [35,
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42, 48], and it was predicted that the 5´ portion of MLL fusion proteins—commonly retained in both wild-type MLL and MLL fusion proteins—is necessary and sufficient for recognition of the target genes (Figure 2A). The ability of MLL fusion proteins to
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activate gene expression and transform hematopoietic progenitors can be evaluated by the myeloid progenitor transformation assay [16]. In this assay, functional MLL fusion
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proteins constitutively activate HOXA9 expression in myeloid progenitors derived from
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murine bone marrow. As a result, the MLL fusion-transduced progenitors expand indefinitely in semi-solid media. Structure/function analysis using the myeloid progenitor transformation assay revealed that the MENIN binding motif and the CXXC domain are required for gene activation by MLL fusion proteins [49-51]. The MLL fusion/MENIN complex further associates with the lens epithelium-derived growth
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factor (LEDGF also known as PSIP1) [52, 53]. LEDGF binds to nucleosomes through
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its PWWP domain [54]. The PWWP and CXXC domains constitute the minimum targeting module (MTM) sufficient for target recognition by MLL fusion proteins (Figure 2A). Accordingly, an artificial protein in which MTM is fused with ENL
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activated HOXA9 expression and immortalized myeloid progenitors [55].
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The PWWP domain of LEDGF specifically binds di-/tri-methylated histone H3 lysine 36 (H3K36me2/3) in vitro [55-57]. Consequently, the nucleosomes that co-precipitated with the PWWP domain contained high levels of H3K36me2/3
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modifications [55]. H3K36me2 is a relatively abundant chromatin modification and slightly enriched in the promoter-proximal region of transcriptionally active genes [57,
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58], while H3K36me3 is located in the gene body region [55, 59]. The CXXC domain
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of MLL binds to non-methylated CpGs, which are clustered in non-silenced promoter regions [60-62]. An artificial protein constituting MTM in which the PWWP domain is fused to the CXXC domain stably binds to the promoter regions of MLL target genes such as HOXA9 [55]. Because H3K36me2 is more abundant at the promoter proximal regions than H3K36me3 [55, 57, 58], the nucleosomes pulled down by MTM are highly
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enriched with H3K36me2 [55]. ASH1L, a histone methylatransferase which generates
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H3K36me2 marks, appear to play a major role in creating the MLL target chromatin [57]. Two points of contact through the PWWP domain and the CXXC domain are necessary for stable target binding [55], indicating that the MLL fusion protein complex
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associates with its target chromatin through both H3K36me2/3 marks and
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non-methylated CpGs. Indeed, the H3K36me2/3 binding mediated by LEDGF is required for the formation of a higher order MLL fusion complex on the target chromatin [10, 52, 57]. However, it should be noted that the MLL fusion protein per se
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may be able to bind the target chromatin without LEDGF [57]. This suggests that the association between the CXXC domain and non-methylated CpGs is the major
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determinant for target recognition, while the association between LEDGF and
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H3K36me2/3 marks is for the reinforcement of the interaction with the target chromatin. Chromatin immunoprecipitation followed by deep sequencing showed that the MLL-AF6
fusion
protein
co-localized
with
non-methylated
CpGs
in
the
promoter-proximal region [55]. Taken together, the MLL fusion complex targets the promoter-proximal regions of transcriptionally active CpG-rich genes, in which
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H3K36me2/3 marks are abundant (Figure 2B). The MLL fusion complex therefore
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represents a piece of transcriptional machinery that re-activates previously active CpG-rich genes.
Additional interactions may be involved in the targeting mechanisms of MLL
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fusion proteins. It has been reported that association with the PAF1 complex is required
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for target recognition [63, 64] (Figure 2A). MENIN and LEDGF bind DNA in a sequence-independent manner [56, 65]. It has been reported that the presence of wild-type MLL, but not its HMT activity, is required for targeting of some MLL fusion
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proteins [63, 66, 67]. These additional interactions/factors likely contribute to proper targeting of MLL fusion proteins. Recently, it was reported that MLL fusion proteins
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also target regions near the transcription end sites of a subset of target genes [68], which
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may involve some of these additional interaction networks. MLL retains multiple PHD fingers, one of which binds to di-/tri-methylated histone H3K4 (H3K4me2/3) [69] (Figure 2A). An artificial construct composed of the CXXC domain and the PHD finger was shown to target the HOXA9 locus in murine embryonic fibroblasts, suggestive of a MENIN-independent targeting mechanism for wild-type MLL [63], which further
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corroborates the idea that the expression of a subset of MLL target genes does not
of the MLL target genes through MENIN (Figure 2B).
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depend on MENIN [48, 70]. Thus, it is likely that MLL fusion proteins target only a part
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Mechanisms of constitutive activation by MLL-AEP fusion proteins
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Transcriptional activation by MLL fusion proteins is mediated through the fusion partner portion. Among more than 70 fusion partners of MLL [9], members of the AF4 gene family, which is composed of AF4 (also known as AFF1), AF5Q31 (also
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known as AFF4), LAF4 (also known as AFF3), and FMR2 (also known as AFF2) [71], are the most frequent fusion partners. The AF4 family protein associates with the
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positive transcription elongation factor b (P-TEFb) complex through the N-terminal
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homology domain (NHD) and ELL family proteins through the ALF domain [10, 72-75] (Figure 3A), both of which facilitate transcription elongation [76, 77]. The AF4 protein complex activates transcriptional elongation in various biological processes such as heat shock response [74] and viral genome transcription of the human immunodeficiency virus (HIV) [78, 79].
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The AF4 family protein associates with the ENL family protein through the
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ANC1 homology domain (AHD) at its C-terminus [80] (Figure 3A). The ENL gene family is composed of ENL (also known as MLLT1) and AF9 (also known as MLLT3), and is also one of the most frequent fusion partners in MLL-r leukemia [9]. The ENL
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family proteins retain a YEATS domain at the N-terminus, which associates with the
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acetylated histone H3 lysine 9/18/27 [81]. Biochemically stable AF4 complexes containing ENL family proteins and P-TEFb have been purified independently by multiple groups [10, 74, 78, 79] and are known by various aliases including AEP and
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SEC (Super Elongation Complex).
MLL-AEP fusion proteins such as MLL-ENL and MLL-AF5Q31 exhibit
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transforming ability in myeloid progenitor transformation assays [10]. An artificial
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protein in which MTM is fused with AHD of ENL or the C-terminal homology domain (CHD) of AF5Q31 can transform myeloid progenitors [82] (Figure 3A). Because both AHD and CHD are the binding platforms for AF4, AF4 was thought to be responsible for conferring transforming ability to the MLL fusion proteins. Because of known transcriptional elongation functions of P-TEFb [74, 78, 79], it was presumed that the
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recruitment of P-TEFb through AF4 triggers the expression of MLL target genes [83,
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84]. Relatively high sensitivities of MLL leukemia cells to CDK9 inhibitors supported this idea [83]. However, structure/function analysis data that support this notion have been missing. Moreover, artificial MLL fusion proteins tethered to the binding platform
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for P-TEFb did not exhibit transcriptional activation activity of Hoxa9 nor transforming
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ability of hematopoietic progenitors [10], suggesting that the recruitment of P-TEFb was not sufficient for the transforming property of MLL fusion proteins. To further identify the essential function(s) of AF4 for transformation, the transforming ability of
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various constructs in which MTM was fused to a subdivided domain of AF4 was assessed (Figure 3A). Among all the AF4 subdomains tested, only the pSER domain
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(retained in the AF4-2C portion) conferred transforming ability [82].
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Biochemical analysis of the associating factors for the pSER domain identified selectivity factor 1 (SL1) as a specific pSER domain binder (Figure 3A). SL1 is a protein complex composed of the TATA-binding protein (TBP) and four TBP-associated factor RNA polymerase I subunits (TAF1A/TAFI48, TAF1B/TAFI63, TAF1C/TAFI110, and TAF1D/TAFI41). SL1 is known as a core component of the
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pre-initiation complex (PIC) of RNA polymerase I (RNAP1) [86-89]. Upstream binding
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factor recruits SL1 onto the promoters of ribosomal RNA genes to initiate RNAP1-dependent transcription [90]. In AEP-dependent gene activation, AF4 recruits SL1 to load TBP onto the TATA element to activate transcription initiation of RNA
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Polymerase II (RNAP2) [82]. Structure/function analysis using a myeloid progenitor
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transformation assay indicated that MLL-AEP fusion proteins aberrantly activate transcription by utilizing the TBP-loading function, but that the recruitment of P-TEFb or ELL elongation factors does not confer transforming ability (Figure 3A). Thus, TBP
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loading, but not transcription elongation, appears to be the rate-limiting step of gene activation of MLL target genes.
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AF4 family proteins also associate with the mediator complex via MED26
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[91]. The mediator complex facilitates the PIC formation of RNAP2 [92]. MED26 binds to the pSER domain of AF4 [85], which can be divided into three subdomains (Figure 3B). Each subdomain contains an evolutionarily conserved motif: the DLXLS motif, the SDE motif, and the NKW motif. MED26 binds to the DLXLS motif, the SDE motif is responsible for binding to SL1, and the NKW motif is required for SL1-mediated
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transcriptional activation, presumably by loading TBP to the TATA element [82, 85].
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While the artificial MTM-pSER fusion protein constitutively activates HOXA9 expression to immortalize hematopoietic progenitors, deletion of either the SDE motif or the NKW motif resulted in loss of transforming ability (Figure 3B). Deletion of the
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DLXLS motif, however, did not abolish transforming ability, indicating that MED26
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recruitment via the pSER domain is dispensable for MLL-AEP-dependent gene activation. Thus, the rate-limiting step of MLL-AEP-dependent gene activation seems
mediated by SL1.
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not to be mediator recruitment via the pSER domain, but appears to be TBP loading
Finally, I propose a model of MLL-AEP-dependent gene activation. The
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minimum domains required for constitutive gene activation are MTM and the SDE and
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NKW motifs of AF4, which induce TBP loading at previously active CpG-rich promoters through SL1 [55, 82, 85]. In my working model (Figure 4), the MLL-AEP fusion protein complex first recognizes the target chromatin containing H3K36me2/3 and non-methylated CpGs. Upon MED26 association with the DLXLS motif of AF4, the mediator complex is recruited to the target chromatin. SL1 binds to the SDE motif
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of AF4 and loads TBP via the NKW motif. The MED26-bound mediator complex likely
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dissociates from the DLXLS motif upon SL1 binding as MED26 does not stably associate with the SL1-bound AF4 complex [85]. After TBP loading, the mediator complex facilitates PIC formation of RNAP2. Lastly, the P-TEFb complex (or ELL
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family proteins) activates transcription elongation. The domains that recruit elongation
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activities and the mediator activity were dispensable for gene activation in hematopoietic progenitors [82, 85], suggesting that recruitment of transcription elongation factors and the mediator complex may be compensated via alternative
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mechanisms through other endogenous factors. Thus, the essential activity provided by MLL-AEP fusion proteins is the TBP loading activity through SL1. This view is quite
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different from the prevailing views, in which recruitment of elongation factors is the
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key function of MLL-AEP fusion proteins [83, 84].
Concluding remarks
In this review article, I review the mechanism of gene activation by MLL-AEP fusion proteins and propose a model in which TBP loading through SL1 is
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the rate-limiting step regulated by MLL-AEP fusion proteins. It should be noted,
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however, that other important functions are mediated by MLL fusion proteins. AHD recruits not only AF4 but also DOT1-like histone H3K79 methyltransferase (DOT1L) [10, 80, 93]. DOT1L is an epigenetic modifier that produces mono-, di-, and
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tri-methylation of histone H3 lysine 79 (H3K79me1/2/3) [94, 95], thereby maintaining a
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protective chromatin environment against SIRT1-dependent gene silencing [96]. An MLL-AF9 mutant carrying L504P/D505P substitutions, which has a reduced binding capacity to AF4 family proteins but a normal binding capacity to DOT1L, failed to
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transform hematopoietic progenitors [97], suggesting that AF4 recruitment is critical for transformation. Genetic ablation of DOT1L, however, resulted in the loss of MLL-AF9
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transforming ability [98-101], indicating that the role of DOT1L is also essential. Thus,
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selective inhibitors of DOT1L HMT have been developed and have exhibited efficacy against MLL-AEP leukemia [102, 103]. Compounds that specifically inhibit the formation of the MLL fusion/MENIN protein
complex have also shown efficacy in
disease models [104, 105], providing hope for improving the prognosis for MLL-r leukemia patients. I consider the TBP loading process via SL1 to be as another potential
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therapeutic target; its inhibition by specific compounds in the future may complement
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the above-mentioned molecularly targeted drug candidates. The molecular mechanisms of MLL fusion-dependent gene activation are still under debate. The proposed model emphasizes the importance of transcription initiation
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mediated by SL1, providing a flesh perspective. It is unknown why MLL pairs up with
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AEP components much more often than with other fusion partners. The AEP-SL1 axis may confer some unaccounted advantages in gene expression. Mechanisms of gene activation by non-AEP fusion partners remain elusive. Hopefully those questions will be
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disease.
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answered in near future to help us understand the molecular basis underlying this
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Acknowledgements
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This work was supported by a JSPS KAKENNHI grant to A.Y. (16H05337). A.Y. receives research funding from Dainippon Sumitomo Pharma Co., Ltd. I thank Dr.
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Satoshi Takahashi for critical reading.
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References [1]
Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of
Drosophila trithorax by 11q23 chromosomal translocations in acute [2]
RI PT
leukemias. Cell. 1992;71:691-700.
Gu Y, Nakamura T, Alder H, et al. The t(4;11) chromosome
translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell. 1992;71:701-708.
Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans GA. A
SC
[3]
trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat Genet. 1992;2:113-118. Krivtsov
AV,
Armstrong
SA.
MLL
translocations,
M AN U
[4]
histone
modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7:823-833. [5]
Li BE, Ernst P. Two decades of leukemia oncoprotein epistasis: the
MLL1 paradigm for epigenetic deregulation in leukemia. Exp Hematol. 2014;42:995-1012.
Yokoyama A. Molecular mechanisms of MLL-associated leukemia.
TE D
[6]
International journal of hematology. 2015;101:352-361. [7]
Dimartino JF, Cleary ML. Mll rearrangements in haematological
malignancies: lessons from clinical and biological studies. Br J Haematol.
EP
1999;106:614-626. [8]
Nagayama J, Tomizawa D, Koh K, et al. Infants with acute
AC C
lymphoblastic leukemia and a germline MLL gene are highly curable with use of chemotherapy alone: results from the Japan Infant Leukemia Study Group. Blood. 2006;107:4663-4665. [9]
Meyer C, Hofmann J, Burmeister T, et al. The MLL recombinome of
acute leukemias in 2013. Leukemia. 2013;27:2165-2176. [10]
Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML. A
higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell. 2010;17:198-212. [11]
Downing JR, Head DR, Raimondi SC, et al. The der(11)-encoded 21
ACCEPTED MANUSCRIPT
MLL/AF-4
fusion
transcript
t(4;11)(q21;q23)-containing
is
acute
consistently
lymphoblastic
detected
leukemia.
in Blood.
1994;83:330-335. [12]
Bursen A, Schwabe K, Ruster B, et al. The AF4.MLL fusion protein
RI PT
is capable of inducing ALL in mice without requirement of MLL.AF4. Blood. 2010;115:3570-3579. [13]
Andersson AK, Ma J, Wang J, et al. The landscape of somatic
mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nat [14]
SC
Genet. 2015;47:330-337.
Forster A, Pannell R, Drynan LF, et al. Engineering de novo
reciprocal chromosomal translocations associated with Mll to replicate [15]
M AN U
primary events of human cancer. Cancer Cell. 2003;3:449-458. Krivtsov AV, Feng Z, Lemieux ME, et al. H3K79 methylation
profiles define murine and human MLL-AF4 leukemias. Cancer Cell. 2008;14:355-368. [16]
Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and
leukemic transformation of a myelomonocytic precursor by retrovirally [17]
TE D
transduced HRX-ENL. Embo J. 1997;16:4226-4237. Krivtsov AV, Twomey D, Feng Z, et al. Transformation from
committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006;442:818-822.
Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM,
EP
[18]
Sauvageau G. Hoxa9 transforms primary bone marrow cells through specific
AC C
collaboration with Meis1a but not Pbx1b. Embo J. 1998;17:3714-3725. [19]
Schnabel
CA,
Jacobs
Y,
Cleary
ML.
HoxA9-mediated
immortalization of myeloid progenitors requires functional interactions with TALE cofactors Pbx and Meis. Oncogene. 2000;19:608-616. [20]
Lawrence HJ, Christensen J, Fong S, et al. Loss of expression of the
Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood. 2005;106:3988-3994. [21]
Lawrence HJ, Helgason CD, Sauvageau G, et al. Mice bearing a
targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood. 1997;89:1922-1930. 22
ACCEPTED MANUSCRIPT
[22]
Armstrong
SA,
Staunton
JE,
Silverman
LB,
et
al.
MLL
translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002;30:41-47. survival
Faber J, Krivtsov AV, Stubbs MC, et al. HOXA9 is required for in
human
MLL-rearranged
acute
2009;113:2375-2385. [24]
leukemias.
Blood.
RI PT
[23]
Wong P, Iwasaki M, Somervaille TC, So CW, Cleary ML. Meis1 is an
essential and rate-limiting regulator of MLL leukemia stem cell potential. [25]
SC
Genes Dev. 2007;21:2762-2774.
Arai S, Yoshimi A, Shimabe M, et al. Evi-1 is a transcriptional
target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells. [26]
Ono
R,
Masuya
MLL-fusion-mediated
M AN U
Blood. 2011;117:6304-6314.
M,
Nakajima
leukemogenesis
H,
et
specifically
al.
Plzf
in
drives
long-term
hematopoietic stem cells. Blood. 2013;122:1271-1283. [27]
Zhang Y, Owens K, Hatem L, et al. Essential role of PR-domain
protein MDS1-EVI1 in MLL-AF9 leukemia. Blood. 2013;122:2888-2892. Placke T, Faber K, Nonami A, et al. Requirement for CDK6 in
TE D
[28]
MLL-rearranged acute myeloid leukemia. Blood. 2014;124:13-23. [29]
van der Linden MH, Willekes M, van Roon E, et al. MLL
fusion-driven
activation
of
CDK6
potentiates
proliferation
in
[30]
EP
MLL-rearranged infant ALL. Cell cycle. 2014;13:834-844. Laurenti E, Frelin C, Xie S, et al. CDK6 levels regulate quiescence
AC C
exit in human hematopoietic stem cells. Cell Stem Cell. 2015;16:302-313. [31]
Ayton PM, Cleary ML. Transformation of myeloid progenitors by
MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev. 2003;17:2298-2307. [32]
Milne TA, Briggs SD, Brock HW, et al. MLL targets SET domain
methyltransferase
activity
to
Hox
gene
promoters.
Molecular
cell.
2002;10:1107-1117. [33]
Nakamura T, Mori T, Tada S, et al. ALL-1 is a histone
methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Molecular cell. 2002;10:1119-1128. 23
ACCEPTED MANUSCRIPT
[34]
Yu BD, Hess JL, Horning SE, Brown GA, Korsmeyer SJ. Altered
Hox expression and segmental identity in Mll-mutant mice. Nature. 1995;378:505-508. [35]
Yagi H, Deguchi K, Aono A, Tani Y, Kishimoto T, Komori T. Growth
RI PT
disturbance in fetal liver hematopoiesis of Mll-mutant mice. Blood. 1998;92:108-117. [36]
Sedkov Y, Tillib S, Mizrokhi L, Mazo A. The bithorax complex is
regulated by trithorax earlier during Drosophila embryogenesis than is the
SC
Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early trithorax transcripts. Development. 1994;120:1907-1917. [37]
Breen TR, Harte PJ. Trithorax regulates multiple homeotic genes in
M AN U
the bithorax and Antennapedia complexes and exerts different tissue-specific, parasegment-specific and promoter-specific effects on each. Development. 1993;117:119-134. [38]
Deschamps J, van Nes J. Developmental regulation of the Hox
genes
during
axial
morphogenesis
2005;132:2931-2942.
the
mouse.
Development.
Wang KC, Helms JA, Chang HY. Regeneration, repair and
TE D
[39]
in
remembering identity: the three Rs of Hox gene expression. Trends in cell biology. 2009;19:268-275.
Yu BD, Hanson RD, Hess JL, Horning SE, Korsmeyer SJ. MLL, a
mammalian
trithorax-group
EP
[40]
gene,
functions
as
a
transcriptional
maintenance factor in morphogenesis. Proc Natl Acad Sci U S A.
AC C
1998;95:10632-10636. [41]
Blobel GA, Kadauke S, Wang E, et al. A reconfigured pattern of
MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit. Molecular cell. 2009;36:970-983. [42]
Jude CD, Climer L, Xu D, Artinger E, Fisher JK, Ernst P. Unique
and independent roles for MLL in adult hematopoietic stem cells and progenitors. Cell Stem Cell. 2007;1:324-337. [43]
Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM,
Weissman IL. Differential expression of novel potential regulators in hematopoietic stem cells. PLoS Genet. 2005;1:e28. 24
ACCEPTED MANUSCRIPT
[44]
Yokoyama A, Ficara F, Murphy MJ, et al. MLL becomes functional
through intra-molecular interaction not by proteolytic processing. PloS one. 2013;8:e73649. [45]
Somervaille TC, Cleary ML. Identification and characterization of
RI PT
leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 2006;10:257-268. [46]
Yokoyama A, Ficara F, Murphy MJ, et al. Proteolytically cleaved
MLL subunits are susceptible to distinct degradation pathways. J Cell Sci. [47]
SC
2011;124:2208-2219.
McMahon KA, Hiew SY, Hadjur S, et al. Mll has a critical role in
fetal and adult hematopoietic stem cell self-renewal. Cell Stem Cell. [48]
M AN U
2007;1:338-345.
Artinger EL, Ernst P. Cell context in the control of self-renewal and
proliferation regulated by MLL1. Cell cycle. 2013;12:2969-2972. [49]
Slany RK, Lavau C, Cleary ML. The oncogenic capacity of
HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol Cell Biol. 1998;18:122-129. Ayton PM, Chen EH, Cleary ML. Binding to nonmethylated CpG
TE D
[50]
DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein. Mol Cell Biol. 2004;24:10470-10478. [51]
Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O,
oncogenic
EP
Meyerson M, Cleary ML. The menin tumor suppressor protein is an essential cofactor
for
MLL-associated
leukemogenesis.
Cell.
AC C
2005;123:207-218. [52]
Yokoyama A, Cleary ML. Menin critically links MLL proteins with
LEDGF on cancer-associated target genes. Cancer Cell. 2008;14:36-46. [53]
Huang J, Gurung B, Wan B, et al. The same pocket in menin binds
both MLL and JUND but has opposite effects on transcription. Nature. 2012;482:542-546. [54]
Botbol Y, Raghavendra NK, Rahman S, Engelman A, Lavigne M.
Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro. Nucleic Acids Res. 2008. [55]
Okuda H, Kawaguchi M, Kanai A, et al. MLL fusion proteins link 25
ACCEPTED MANUSCRIPT
transcriptional coactivators to previously active CpG-rich promoters. Nucleic Acids Res. 2014;42:4241-4256. [56]
Eidahl JO, Crowe BL, North JA, et al. Structural basis for
high-affinity binding of LEDGF PWWP to mononucleosomes. Nucleic Acids [57]
RI PT
Res. 2013;41:3924-3936.
Zhu L, Li Q, Wong SH, et al. ASH1L Links Histone H3 Lysine 36
Dimethylation to MLL Leukemia. Cancer Discov. 2016;6:770-783. [58]
Kuo AJ, Cheung P, Chen K, et al. NSD2 links dimethylation of
SC
histone H3 at lysine 36 to oncogenic programming. Molecular cell. 2011;44:609-620. [59]
Barski A, Cuddapah S, Cui K, et al. High-resolution profiling of
[60]
M AN U
histone methylations in the human genome. Cell. 2007;129:823-837. Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K, Titgemeyer F,
Slany RK. The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation. Nucleic Acids Res. 2002;30:958-965. [61]
Allen MD, Grummitt CG, Hilcenko C, et al. Solution structure of the
TE D
nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase. Embo J. 2006;25:4503-4512. [62]
Cierpicki T, Risner LE, Grembecka J, et al. Structure of the MLL
CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. [63]
EP
Nat Struct Mol Biol. 2010;17:62-68. Milne TA, Kim J, Wang GG, et al. Multiple interactions recruit
AC C
MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis. Molecular cell. 2010;38:853-863. [64]
Muntean AG, Tan J, Sitwala K, et al. The PAF complex synergizes
with MLL fusion proteins at HOX loci to promote leukemogenesis. Cancer Cell. 2010;17:609-621. [65]
La P, Silva AC, Hou Z, et al. Direct binding of DNA by tumor
suppressor menin. J Biol Chem. 2004;279:49045-49054. [66]
Thiel
AT,
Blessington
P,
Zou
T,
et
al.
MLL-AF9-induced
leukemogenesis requires coexpression of the wild-type Mll allele. Cancer Cell. 2010;17:148-159. 26
ACCEPTED MANUSCRIPT
[67]
Mishra BP, Zaffuto KM, Artinger EL, et al. The histone
methyltransferase activity of MLL1 is dispensable for hematopoiesis and leukemogenesis. Cell reports. 2014;7:1239-1247. [68]
Garcia-Cuellar MP, Buttner C, Bartenhagen C, Dugas M, Slany RK.
RI PT
Leukemogenic MLL-ENL Fusions Induce Alternative Chromatin States to Drive a Functionally Dichotomous Group of Target Genes. Cell reports. 2016;15:310-322. [69]
Wang Z, Song J, Milne TA, et al. Pro isomerization in MLL1 cassette
connects
H3K4me
readout
to
CyP33
and
SC
PHD3-bromo
HDAC-mediated repression. Cell. 2010;141:1183-1194. [70]
Li BE, Gan T, Meyerson M, Rabbitts TH, Ernst P. Distinct pathways
M AN U
regulated by menin and by MLL1 in hematopoietic stem cells and developing B cells. Blood. 2013;122:2039-2046. [71]
Nilson I, Reichel M, Ennas MG, et al. Exon/intron structure of the
human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia. Br J Haematol. 1997;98:157-169.
Estable MC, Naghavi MH, Kato H, et al. MCEF, the newest member
TE D
[72]
of the AF4 family of transcription factors involved in leukemia, is a positive transcription
elongation
2002;9:234-245.
protein.
J
Biomed
Sci.
Benedikt A, Baltruschat S, Scholz B, et al. The leukemogenic
EP
[73]
factor-b-associated
AF4-MLL fusion protein causes P-TEFb kinase activation and altered
AC C
epigenetic signatures. Leukemia. 2011;25:135-144. [74]
Lin C, Smith ER, Takahashi H, et al. AFF4, a component of the
ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Molecular cell. 2010;37:429-437. [75]
Bitoun E, Oliver PL, Davies KE. The mixed-lineage leukemia fusion
partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates
coordinated
chromatin
remodeling.
Hum
Mol
Genet.
2007;16:92-106. [76]
Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway JW.
An RNA polymerase II elongation factor encoded by the human ELL gene. 27
ACCEPTED MANUSCRIPT
Science. 1996;271:1873-1876. [77]
Peterlin BM, Price DH. Controlling the elongation phase of
transcription with P-TEFb. Molecular cell. 2006;23:297-305. [78]
Sobhian B, Laguette N, Yatim A, et al. HIV-1 Tat assembles a
the 7SK snRNP. Molecular cell. 2010;38:439-451. [79]
RI PT
multifunctional transcription elongation complex and stably associates with He N, Liu M, Hsu J, et al. HIV-1 Tat and host AFF4 recruit two
transcription elongation factors into a bifunctional complex for coordinated [80]
SC
activation of HIV-1 transcription. Molecular cell. 2010;38:428-438.
Mueller D, Bach C, Zeisig D, et al. A role for the MLL fusion partner
2007;110:4445-4454. [81]
M AN U
ENL in transcriptional elongation and chromatin modification. Blood. Li Y, Wen H, Xi Y, et al. AF9 YEATS domain links histone
acetylation to DOT1L-mediated H3K79 methylation. Cell. 2014;159:558-571. [82]
Okuda H, Kanai A, Ito S, Matsui H, Yokoyama A. AF4 uses the SL1
components
of
RNAP1
machinery
to
initiate
MLL
fusion-
and
AEP-dependent transcription. Nat Commun. 2015;6:8869. Mueller D, Garcia-Cuellar MP, Bach C, Buhl S, Maethner E, Slany
TE D
[83]
RK. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 2009;7:e1000249. [84]
Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a
EP
molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer. 2010;10:721-728.
Okuda H, Takahashi S, Takaori-Kondo A, Yokoyama A. TBP loading
AC C
[85]
by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription. Cell cycle. In press. [86]
Learned RM, Cordes S, Tjian R. Purification and characterization of
a transcription factor that confers promoter specificity to human RNA polymerase I. Mol Cell Biol. 1985;5:1358-1369. [87]
Comai L, Zomerdijk JC, Beckmann H, Zhou S, Admon A, Tjian R.
Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits. Science. 1994;266:1966-1972. [88]
Eberhard D, Tora L, Egly JM, Grummt I. A TBP-containing 28
ACCEPTED MANUSCRIPT
multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I. Nucleic Acids Res. 1993;21:4180-4186. of
Gorski JJ, Pathak S, Panov K, et al. A novel TBP-associated factor SL1
functions
in
RNA polymerase
I
transcription.
2007;26:1560-1568. [90]
Goodfellow
SJ,
Zomerdijk
JC.
Basic
EMBO
J.
RI PT
[89]
mechanisms
in
RNA
polymerase I transcription of the ribosomal RNA genes. Sub-cellular biochemistry. 2013;61:211-236.
Takahashi H, Parmely TJ, Sato S, et al. Human mediator subunit
SC
[91]
MED26 functions as a docking site for transcription elongation factors. Cell. 2011;146:92-104.
Malik S, Roeder RG. The metazoan Mediator co-activator complex
M AN U
[92]
as an integrative hub for transcriptional regulation. Nat Rev Genet. 2010;11:761-772. [93]
Kuntimaddi A, Achille NJ, Thorpe J, et al. Degree of Recruitment of
DOT1L to MLL-AF9 Defines Level of H3K79 Di- and Tri-methylation on Target Genes and Transformation Potential. Cell reports. 2015;11:808-820. Feng Q, Wang H, Ng HH, et al. Methylation of H3-lysine 79 is
TE D
[94]
mediated by a new family of HMTases without a SET domain. Curr Biol. 2002;12:1052-1058. [95]
Jones B, Su H, Bhat A, et al. The histone H3K79 methyltransferase
EP
Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet. 2008;4:e1000190. Chen
CW,
AC C
[96]
Koche
RP,
Sinha
AU,
et
al.
DOT1L inhibits
SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nat Med. 2015;21:335-343. [97]
Biswas D, Milne TA, Basrur V, et al. Function of leukemogenic
mixed lineage leukemia 1 (MLL) fusion proteins through distinct partner protein complexes. Proc Natl Acad Sci U S A. 2011;108:15751-15756. [98]
Bernt KM, Zhu N, Sinha AU, et al. MLL-rearranged leukemia is
dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell. 2011;20:66-78. [99]
Chang MJ, Wu H, Achille NJ, et al. Histone H3 lysine 79 29
ACCEPTED MANUSCRIPT
methyltransferase Dot1 is required for immortalization by MLL oncogenes. Cancer Res. 2010;70:10234-10242. [100]
Nguyen AT, Taranova O, He J, Zhang Y. DOT1L, the H3K79
Blood. 2011;117:6912-6922. [101]
RI PT
methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Jo SY, Granowicz EM, Maillard I, Thomas D, Hess JL. Requirement
for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation. Blood. 2011;117:4759-4768.
Daigle SR, Olhava EJ, Therkelsen CA, et al. Selective killing of
SC
[102]
mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011;20:53-65.
Daigle SR, Olhava EJ, Therkelsen CA, et al. Potent inhibition of
M AN U
[103]
DOT1L as treatment of MLL-fusion leukemia. Blood. 2013;122:1017-1025. [104]
Grembecka J, He S, Shi A, et al. Menin-MLL inhibitors reverse
oncogenic activity of MLL fusion proteins in leukemia. Nature chemical biology. 2012;8:277-284. [105]
Borkin D, He S, Miao H, et al. Pharmacologic Inhibition of the
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Menin-MLL Interaction Blocks Progression of MLL Leukemia In Vivo.
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Cancer Cell. 2015;27:589-602.
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Figure legends
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FIGURE 1 Models of gene activation by MLL and MLL fusion complexes. Expression of
self-renewal-related genes in MLL-r leukemia (A) and normal hematopoiesis (B). The cartoon
The mechanisms of target recognition by MLL and MLL fusion complexes.
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FIGURE 2.
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was modified from a previous review article [6]
(A) Schematic structures of MLL and MLL fusion complexes. The domain structures
responsible for various protein-protein interactions are indicated. hMBM: high affinity menin
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binding motif; LBD: LEDGF binding domain; PHD: plant homeodomain; HBM; HCF binding
motif; AD: activation domain; PS: processing site; Win: WDR5 interaction motif; IBD:
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Integrase binding domain; MTM: minimum targeting module. The cartoon was modified from a
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previous review article [6]. (B) Schematic representation of the mechanisms by which MLL and
MLL fusion complexes recognize their target chromatin.
FIGURE 3.
The domains/functions required for gene activation by MLL-AEP fusion
proteins. (A) The functions of various domains of AF4 are depicted schematically. The abilities
to transform myeloid progenitors and activate Hoxa9 expression in the form of MTM fusion are
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indicated along with various functions. The ALF and pSER domains were originally defined by
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Nilson et al. [71]. Their definition was slightly modified as the DLXLS motif, which was
originally included in the ALF domain, was included in the pSER domain in our reports [82, 85].
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(B) The functions of various subdomains in the pSER domain are depicted schematically.
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FIGURE 4. Model of gene activation by MLL-AEP fusion proteins. The rate-limiting step
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carried out by MLL-AEP fusion proteins is highlighted in red.
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Highlights
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• • •
MLL gene rearrangements cause acute leukemia by constitutive activation of self-renewal-related genes MLL fusion proteins target the promoter proximal regions of previously-active CpG-rich genes TBP loading through SL1 is the rate-limiting step regulated by MLL-AEP fusion proteins. Recruitment of transcription elongation and mediator activities by MLL fusion proteins is not essential for leukemogenesis.
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•
S0301-472X(16)30697-X
DOI:
10.1016/j.exphem.2016.10.014
Reference:
EXPHEM 3484
To appear in:
Experimental Hematology
Received Date: 12 August 2016 Revised Date:
14 October 2016
Accepted Date: 29 October 2016
Please cite this article as: Yokoyama A, Transcriptional activation by MLL fusion proteins in leukemogenesis, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.10.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Review Article
Department of Hematology and Oncology, Graduate School of Medicine, 54
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1
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Akihiko Yokoyama1, 2
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Transcriptional activation by MLL fusion proteins in leukemogenesis
Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Division of Hematological Malignancy, National Cancer Center Research Institute,
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Tokyo 104-0045, Japan
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Address correspondence to: Akihiko Yokoyama Ph.D.
[email protected] Tel: 81-75-751-3096 Fax: 81-75-751-3201
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Category for Table of Contents: Malignant Hematopoiesis
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Word count: 3,099
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Abstract
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Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause aggressive leukemia. Fusion proteins of MLL and a component of the AF4 family/ENL family/P-TEFb complex (AEP) are responsible for two-thirds of MLL-associated
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leukemia cases. MLL-AEP fusion proteins trigger aberrant self-renewal of
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hematopoietic progenitors by constitutively activating self-renewal-related genes. MLL-AEP fusion proteins activate transcription initiation by loading the TATA-binding protein (TBP) to the TATA element via selectivity factor 1. Although AEP retains
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transcription elongation and mediator recruiting activities, the rate-limiting step activated by MLL-AEP fusion proteins appears to be the TBP loading step unlike
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prevailing views, in which the recruitment of transcription elongation activities are
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emphasized. Here, I review recent advances towards elucidating the mechanisms underlying gene activation by MLL-AEP fusion proteins in leukemogenesis.
Keywords: MLL, Leukemia, AF4, Transcription, Mediator
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Introduction Chromosomal translocations generate fusion genes of the mixed lineage leukemia
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(MLL) gene (also known as MLL1, HRX, HTRX, KMT2A, and ALL1) [1-3] and various partner genes, causing acute leukemia [4-6]. Leukemia involving MLL gene
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rearrangements (hereafter referred to as MLL-r leukemia) accounts for 5–10% of all
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acute leukemia cases and is often accompanied by an unfavorable prognosis [7, 8]. To date, more than 70 MLL fusion genes have been reported, among which MLL fusions with a component of the AF4 family/ENL family/P-TEFb complex (AEP) constitute
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two-thirds of all MLL-r leukemia cases [9, 10]. Because chromosomal translocations of MLL occur within the introns of MLL and its partner gene X, two types of fusion genes
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are generated: MLL-X and X-MLL. In t(4:11) leukemia cases (fusion of MLL and AF4),
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the AF4-MLL fusion gene is often (not always) expressed [11] and its product exhibits oncogenic properties in mouse models [12]. Nevertheless, the MLL-X fusion gene is transcribed in all MLL-r leukemia cases and its product is thought to be the major contributor for oncogenesis [11]. Hereafter, the MLL-X fusion gene will be referred to as the MLL fusion gene. Compared with other cancers, MLL-r leukemia involves fewer genetic alterations [13]. Many mouse disease models have demonstrated that the
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engineered expression of the MLL fusion gene causes leukemia in a relatively short
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period of time [14-16], supporting the notion that the MLL fusion gene is the major driver of leukemogenesis.
MLL fusion proteins cause constitutive expression of a subset of genes
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including HOXA9 and MEIS1, which are normally expressed specifically in immature
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hematopoietic progenitors such as hematopoietic stem cells (HSCs) [17] (Figure 1 A and B). The expression of HOXA9 facilitates the expansion of immature hematopoietic progenitors [18]. Constitutive expression of HOXA9 causes immortalization of
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immature hematopoietic progenitors ex vivo [19], whereas knockout of HOXA9 compromises the capacity to reconstitute the hematopoietic system [20, 21]. HOXA9 is
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highly expressed in most MLL-r leukemia cells [17, 22] and is required for their
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survival [23]. MEIS1, which is also highly expressed in MLL-r leukemia cells, is a cofactor of HOXA9 whose expression levels correlate with leukemia stem cell potential in vivo [24]. Collaborative functions of HOXA9 and MEIS1 appear to be the major driving force of leukemogenesis as co-expression of both genes efficiently induces leukemia in mouse models [18]. Several other MLL target genes including EVI1 and
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PLZF have also been shown to facilitate oncogenic self-renewal of MLL leukemia cells
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[25-27]. MLL fusion proteins also activate the expression of CDK6 [28, 29], the activity of which is required for the exit of HSCs from quiescence [30]. Taken together, these findings demonstrate that MLL fusion proteins confer an HSC-like self-renewing
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property to hematopoietic progenitors via the constitutive activation of various
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self-renewal-related genes, thereby causing leukemia [18, 19, 31]. Therefore, the focus of this article is the mechanism of constitutive gene activation by a group of MLL-AEP
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fusion proteins.
MLL is a transcriptional maintenance factor of cellular memory genes
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MLL is an ortholog of the Drosophila trithorax protein [1, 2]. Both have an
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evolutionarily conserved SET domain with histone methyl transferase (HMT) activity towards histone H3 lysine 4 [32, 33] (Figure 2A). Knockout of MLL results in impaired posterior homeobox (HOX) gene expression [34, 35], similar to the consequences of the genetic ablation of trithorax in Drosophila [36, 37]. HOX genes are referred to as “cellular memory” genes as their position-specific expression is maintained throughout
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development [38, 39]. MLL is not required for the initial activation of HOX genes, but
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is necessary for their continuous expression during development [40]. Accordingly, MLL is considered a transcriptional maintenance factor of cellular memory. MLL is retained on chromatin during mitosis, such that it can efficiently activate its target genes
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in the next G1 phase [41], thereby maintaining the expression of cellular memory genes
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for multiple cell divisions.
MLL activates the expression of posterior HOXA genes in the hematopoietic cell lineage [35, 42]. Posterior HOXA gene expression is highest at the immature
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progenitor stage (e.g., HSCs and multi-potent progenitors [MPPs]) [43], but progressively declines as cells differentiate, and eventually diminishes at the terminally
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differentiated stages (Figure 1B) [44, 45]. MLL promotes proper expansion of the
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immature hematopoietic compartments of both adult and fetal hematopoietic systems presumably by upregulating posterior HOXA gene expression [35, 42, 46, 47]. The effects of MLL deficiency are prominent in situations where hematopoietic progenitors expand rapidly, such as the reconstitution of hematopoietic system [42, 47]. These results indicate that MLL regulates the proper expansion of immature hematopoietic
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progenitors by maintaining the expression of self-renewal-related genes at immature
Mechanisms of target recognition by MLL proteins
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progenitor stages.
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MLL fusion proteins and wild-type MLL regulate a common set of genes [35,
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42, 48], and it was predicted that the 5´ portion of MLL fusion proteins—commonly retained in both wild-type MLL and MLL fusion proteins—is necessary and sufficient for recognition of the target genes (Figure 2A). The ability of MLL fusion proteins to
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activate gene expression and transform hematopoietic progenitors can be evaluated by the myeloid progenitor transformation assay [16]. In this assay, functional MLL fusion
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proteins constitutively activate HOXA9 expression in myeloid progenitors derived from
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murine bone marrow. As a result, the MLL fusion-transduced progenitors expand indefinitely in semi-solid media. Structure/function analysis using the myeloid progenitor transformation assay revealed that the MENIN binding motif and the CXXC domain are required for gene activation by MLL fusion proteins [49-51]. The MLL fusion/MENIN complex further associates with the lens epithelium-derived growth
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factor (LEDGF also known as PSIP1) [52, 53]. LEDGF binds to nucleosomes through
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its PWWP domain [54]. The PWWP and CXXC domains constitute the minimum targeting module (MTM) sufficient for target recognition by MLL fusion proteins (Figure 2A). Accordingly, an artificial protein in which MTM is fused with ENL
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activated HOXA9 expression and immortalized myeloid progenitors [55].
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The PWWP domain of LEDGF specifically binds di-/tri-methylated histone H3 lysine 36 (H3K36me2/3) in vitro [55-57]. Consequently, the nucleosomes that co-precipitated with the PWWP domain contained high levels of H3K36me2/3
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modifications [55]. H3K36me2 is a relatively abundant chromatin modification and slightly enriched in the promoter-proximal region of transcriptionally active genes [57,
EP
58], while H3K36me3 is located in the gene body region [55, 59]. The CXXC domain
AC C
of MLL binds to non-methylated CpGs, which are clustered in non-silenced promoter regions [60-62]. An artificial protein constituting MTM in which the PWWP domain is fused to the CXXC domain stably binds to the promoter regions of MLL target genes such as HOXA9 [55]. Because H3K36me2 is more abundant at the promoter proximal regions than H3K36me3 [55, 57, 58], the nucleosomes pulled down by MTM are highly
9
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enriched with H3K36me2 [55]. ASH1L, a histone methylatransferase which generates
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H3K36me2 marks, appear to play a major role in creating the MLL target chromatin [57]. Two points of contact through the PWWP domain and the CXXC domain are necessary for stable target binding [55], indicating that the MLL fusion protein complex
SC
associates with its target chromatin through both H3K36me2/3 marks and
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non-methylated CpGs. Indeed, the H3K36me2/3 binding mediated by LEDGF is required for the formation of a higher order MLL fusion complex on the target chromatin [10, 52, 57]. However, it should be noted that the MLL fusion protein per se
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may be able to bind the target chromatin without LEDGF [57]. This suggests that the association between the CXXC domain and non-methylated CpGs is the major
EP
determinant for target recognition, while the association between LEDGF and
AC C
H3K36me2/3 marks is for the reinforcement of the interaction with the target chromatin. Chromatin immunoprecipitation followed by deep sequencing showed that the MLL-AF6
fusion
protein
co-localized
with
non-methylated
CpGs
in
the
promoter-proximal region [55]. Taken together, the MLL fusion complex targets the promoter-proximal regions of transcriptionally active CpG-rich genes, in which
10
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H3K36me2/3 marks are abundant (Figure 2B). The MLL fusion complex therefore
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represents a piece of transcriptional machinery that re-activates previously active CpG-rich genes.
Additional interactions may be involved in the targeting mechanisms of MLL
SC
fusion proteins. It has been reported that association with the PAF1 complex is required
M AN U
for target recognition [63, 64] (Figure 2A). MENIN and LEDGF bind DNA in a sequence-independent manner [56, 65]. It has been reported that the presence of wild-type MLL, but not its HMT activity, is required for targeting of some MLL fusion
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proteins [63, 66, 67]. These additional interactions/factors likely contribute to proper targeting of MLL fusion proteins. Recently, it was reported that MLL fusion proteins
EP
also target regions near the transcription end sites of a subset of target genes [68], which
AC C
may involve some of these additional interaction networks. MLL retains multiple PHD fingers, one of which binds to di-/tri-methylated histone H3K4 (H3K4me2/3) [69] (Figure 2A). An artificial construct composed of the CXXC domain and the PHD finger was shown to target the HOXA9 locus in murine embryonic fibroblasts, suggestive of a MENIN-independent targeting mechanism for wild-type MLL [63], which further
11
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corroborates the idea that the expression of a subset of MLL target genes does not
of the MLL target genes through MENIN (Figure 2B).
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depend on MENIN [48, 70]. Thus, it is likely that MLL fusion proteins target only a part
SC
Mechanisms of constitutive activation by MLL-AEP fusion proteins
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Transcriptional activation by MLL fusion proteins is mediated through the fusion partner portion. Among more than 70 fusion partners of MLL [9], members of the AF4 gene family, which is composed of AF4 (also known as AFF1), AF5Q31 (also
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known as AFF4), LAF4 (also known as AFF3), and FMR2 (also known as AFF2) [71], are the most frequent fusion partners. The AF4 family protein associates with the
EP
positive transcription elongation factor b (P-TEFb) complex through the N-terminal
AC C
homology domain (NHD) and ELL family proteins through the ALF domain [10, 72-75] (Figure 3A), both of which facilitate transcription elongation [76, 77]. The AF4 protein complex activates transcriptional elongation in various biological processes such as heat shock response [74] and viral genome transcription of the human immunodeficiency virus (HIV) [78, 79].
12
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The AF4 family protein associates with the ENL family protein through the
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ANC1 homology domain (AHD) at its C-terminus [80] (Figure 3A). The ENL gene family is composed of ENL (also known as MLLT1) and AF9 (also known as MLLT3), and is also one of the most frequent fusion partners in MLL-r leukemia [9]. The ENL
SC
family proteins retain a YEATS domain at the N-terminus, which associates with the
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acetylated histone H3 lysine 9/18/27 [81]. Biochemically stable AF4 complexes containing ENL family proteins and P-TEFb have been purified independently by multiple groups [10, 74, 78, 79] and are known by various aliases including AEP and
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SEC (Super Elongation Complex).
MLL-AEP fusion proteins such as MLL-ENL and MLL-AF5Q31 exhibit
EP
transforming ability in myeloid progenitor transformation assays [10]. An artificial
AC C
protein in which MTM is fused with AHD of ENL or the C-terminal homology domain (CHD) of AF5Q31 can transform myeloid progenitors [82] (Figure 3A). Because both AHD and CHD are the binding platforms for AF4, AF4 was thought to be responsible for conferring transforming ability to the MLL fusion proteins. Because of known transcriptional elongation functions of P-TEFb [74, 78, 79], it was presumed that the
13
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recruitment of P-TEFb through AF4 triggers the expression of MLL target genes [83,
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84]. Relatively high sensitivities of MLL leukemia cells to CDK9 inhibitors supported this idea [83]. However, structure/function analysis data that support this notion have been missing. Moreover, artificial MLL fusion proteins tethered to the binding platform
SC
for P-TEFb did not exhibit transcriptional activation activity of Hoxa9 nor transforming
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ability of hematopoietic progenitors [10], suggesting that the recruitment of P-TEFb was not sufficient for the transforming property of MLL fusion proteins. To further identify the essential function(s) of AF4 for transformation, the transforming ability of
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various constructs in which MTM was fused to a subdivided domain of AF4 was assessed (Figure 3A). Among all the AF4 subdomains tested, only the pSER domain
EP
(retained in the AF4-2C portion) conferred transforming ability [82].
AC C
Biochemical analysis of the associating factors for the pSER domain identified selectivity factor 1 (SL1) as a specific pSER domain binder (Figure 3A). SL1 is a protein complex composed of the TATA-binding protein (TBP) and four TBP-associated factor RNA polymerase I subunits (TAF1A/TAFI48, TAF1B/TAFI63, TAF1C/TAFI110, and TAF1D/TAFI41). SL1 is known as a core component of the
14
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pre-initiation complex (PIC) of RNA polymerase I (RNAP1) [86-89]. Upstream binding
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factor recruits SL1 onto the promoters of ribosomal RNA genes to initiate RNAP1-dependent transcription [90]. In AEP-dependent gene activation, AF4 recruits SL1 to load TBP onto the TATA element to activate transcription initiation of RNA
SC
Polymerase II (RNAP2) [82]. Structure/function analysis using a myeloid progenitor
M AN U
transformation assay indicated that MLL-AEP fusion proteins aberrantly activate transcription by utilizing the TBP-loading function, but that the recruitment of P-TEFb or ELL elongation factors does not confer transforming ability (Figure 3A). Thus, TBP
TE D
loading, but not transcription elongation, appears to be the rate-limiting step of gene activation of MLL target genes.
EP
AF4 family proteins also associate with the mediator complex via MED26
AC C
[91]. The mediator complex facilitates the PIC formation of RNAP2 [92]. MED26 binds to the pSER domain of AF4 [85], which can be divided into three subdomains (Figure 3B). Each subdomain contains an evolutionarily conserved motif: the DLXLS motif, the SDE motif, and the NKW motif. MED26 binds to the DLXLS motif, the SDE motif is responsible for binding to SL1, and the NKW motif is required for SL1-mediated
15
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transcriptional activation, presumably by loading TBP to the TATA element [82, 85].
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While the artificial MTM-pSER fusion protein constitutively activates HOXA9 expression to immortalize hematopoietic progenitors, deletion of either the SDE motif or the NKW motif resulted in loss of transforming ability (Figure 3B). Deletion of the
SC
DLXLS motif, however, did not abolish transforming ability, indicating that MED26
M AN U
recruitment via the pSER domain is dispensable for MLL-AEP-dependent gene activation. Thus, the rate-limiting step of MLL-AEP-dependent gene activation seems
mediated by SL1.
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not to be mediator recruitment via the pSER domain, but appears to be TBP loading
Finally, I propose a model of MLL-AEP-dependent gene activation. The
EP
minimum domains required for constitutive gene activation are MTM and the SDE and
AC C
NKW motifs of AF4, which induce TBP loading at previously active CpG-rich promoters through SL1 [55, 82, 85]. In my working model (Figure 4), the MLL-AEP fusion protein complex first recognizes the target chromatin containing H3K36me2/3 and non-methylated CpGs. Upon MED26 association with the DLXLS motif of AF4, the mediator complex is recruited to the target chromatin. SL1 binds to the SDE motif
16
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of AF4 and loads TBP via the NKW motif. The MED26-bound mediator complex likely
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dissociates from the DLXLS motif upon SL1 binding as MED26 does not stably associate with the SL1-bound AF4 complex [85]. After TBP loading, the mediator complex facilitates PIC formation of RNAP2. Lastly, the P-TEFb complex (or ELL
SC
family proteins) activates transcription elongation. The domains that recruit elongation
M AN U
activities and the mediator activity were dispensable for gene activation in hematopoietic progenitors [82, 85], suggesting that recruitment of transcription elongation factors and the mediator complex may be compensated via alternative
TE D
mechanisms through other endogenous factors. Thus, the essential activity provided by MLL-AEP fusion proteins is the TBP loading activity through SL1. This view is quite
EP
different from the prevailing views, in which recruitment of elongation factors is the
AC C
key function of MLL-AEP fusion proteins [83, 84].
Concluding remarks
In this review article, I review the mechanism of gene activation by MLL-AEP fusion proteins and propose a model in which TBP loading through SL1 is
17
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the rate-limiting step regulated by MLL-AEP fusion proteins. It should be noted,
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however, that other important functions are mediated by MLL fusion proteins. AHD recruits not only AF4 but also DOT1-like histone H3K79 methyltransferase (DOT1L) [10, 80, 93]. DOT1L is an epigenetic modifier that produces mono-, di-, and
SC
tri-methylation of histone H3 lysine 79 (H3K79me1/2/3) [94, 95], thereby maintaining a
M AN U
protective chromatin environment against SIRT1-dependent gene silencing [96]. An MLL-AF9 mutant carrying L504P/D505P substitutions, which has a reduced binding capacity to AF4 family proteins but a normal binding capacity to DOT1L, failed to
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transform hematopoietic progenitors [97], suggesting that AF4 recruitment is critical for transformation. Genetic ablation of DOT1L, however, resulted in the loss of MLL-AF9
EP
transforming ability [98-101], indicating that the role of DOT1L is also essential. Thus,
AC C
selective inhibitors of DOT1L HMT have been developed and have exhibited efficacy against MLL-AEP leukemia [102, 103]. Compounds that specifically inhibit the formation of the MLL fusion/MENIN protein
complex have also shown efficacy in
disease models [104, 105], providing hope for improving the prognosis for MLL-r leukemia patients. I consider the TBP loading process via SL1 to be as another potential
18
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therapeutic target; its inhibition by specific compounds in the future may complement
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the above-mentioned molecularly targeted drug candidates. The molecular mechanisms of MLL fusion-dependent gene activation are still under debate. The proposed model emphasizes the importance of transcription initiation
SC
mediated by SL1, providing a flesh perspective. It is unknown why MLL pairs up with
M AN U
AEP components much more often than with other fusion partners. The AEP-SL1 axis may confer some unaccounted advantages in gene expression. Mechanisms of gene activation by non-AEP fusion partners remain elusive. Hopefully those questions will be
AC C
EP
disease.
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answered in near future to help us understand the molecular basis underlying this
19
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Acknowledgements
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This work was supported by a JSPS KAKENNHI grant to A.Y. (16H05337). A.Y. receives research funding from Dainippon Sumitomo Pharma Co., Ltd. I thank Dr.
AC C
EP
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M AN U
SC
Satoshi Takahashi for critical reading.
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References [1]
Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of
Drosophila trithorax by 11q23 chromosomal translocations in acute [2]
RI PT
leukemias. Cell. 1992;71:691-700.
Gu Y, Nakamura T, Alder H, et al. The t(4;11) chromosome
translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell. 1992;71:701-708.
Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans GA. A
SC
[3]
trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat Genet. 1992;2:113-118. Krivtsov
AV,
Armstrong
SA.
MLL
translocations,
M AN U
[4]
histone
modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7:823-833. [5]
Li BE, Ernst P. Two decades of leukemia oncoprotein epistasis: the
MLL1 paradigm for epigenetic deregulation in leukemia. Exp Hematol. 2014;42:995-1012.
Yokoyama A. Molecular mechanisms of MLL-associated leukemia.
TE D
[6]
International journal of hematology. 2015;101:352-361. [7]
Dimartino JF, Cleary ML. Mll rearrangements in haematological
malignancies: lessons from clinical and biological studies. Br J Haematol.
EP
1999;106:614-626. [8]
Nagayama J, Tomizawa D, Koh K, et al. Infants with acute
AC C
lymphoblastic leukemia and a germline MLL gene are highly curable with use of chemotherapy alone: results from the Japan Infant Leukemia Study Group. Blood. 2006;107:4663-4665. [9]
Meyer C, Hofmann J, Burmeister T, et al. The MLL recombinome of
acute leukemias in 2013. Leukemia. 2013;27:2165-2176. [10]
Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML. A
higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell. 2010;17:198-212. [11]
Downing JR, Head DR, Raimondi SC, et al. The der(11)-encoded 21
ACCEPTED MANUSCRIPT
MLL/AF-4
fusion
transcript
t(4;11)(q21;q23)-containing
is
acute
consistently
lymphoblastic
detected
leukemia.
in Blood.
1994;83:330-335. [12]
Bursen A, Schwabe K, Ruster B, et al. The AF4.MLL fusion protein
RI PT
is capable of inducing ALL in mice without requirement of MLL.AF4. Blood. 2010;115:3570-3579. [13]
Andersson AK, Ma J, Wang J, et al. The landscape of somatic
mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nat [14]
SC
Genet. 2015;47:330-337.
Forster A, Pannell R, Drynan LF, et al. Engineering de novo
reciprocal chromosomal translocations associated with Mll to replicate [15]
M AN U
primary events of human cancer. Cancer Cell. 2003;3:449-458. Krivtsov AV, Feng Z, Lemieux ME, et al. H3K79 methylation
profiles define murine and human MLL-AF4 leukemias. Cancer Cell. 2008;14:355-368. [16]
Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and
leukemic transformation of a myelomonocytic precursor by retrovirally [17]
TE D
transduced HRX-ENL. Embo J. 1997;16:4226-4237. Krivtsov AV, Twomey D, Feng Z, et al. Transformation from
committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006;442:818-822.
Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM,
EP
[18]
Sauvageau G. Hoxa9 transforms primary bone marrow cells through specific
AC C
collaboration with Meis1a but not Pbx1b. Embo J. 1998;17:3714-3725. [19]
Schnabel
CA,
Jacobs
Y,
Cleary
ML.
HoxA9-mediated
immortalization of myeloid progenitors requires functional interactions with TALE cofactors Pbx and Meis. Oncogene. 2000;19:608-616. [20]
Lawrence HJ, Christensen J, Fong S, et al. Loss of expression of the
Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood. 2005;106:3988-3994. [21]
Lawrence HJ, Helgason CD, Sauvageau G, et al. Mice bearing a
targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood. 1997;89:1922-1930. 22
ACCEPTED MANUSCRIPT
[22]
Armstrong
SA,
Staunton
JE,
Silverman
LB,
et
al.
MLL
translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002;30:41-47. survival
Faber J, Krivtsov AV, Stubbs MC, et al. HOXA9 is required for in
human
MLL-rearranged
acute
2009;113:2375-2385. [24]
leukemias.
Blood.
RI PT
[23]
Wong P, Iwasaki M, Somervaille TC, So CW, Cleary ML. Meis1 is an
essential and rate-limiting regulator of MLL leukemia stem cell potential. [25]
SC
Genes Dev. 2007;21:2762-2774.
Arai S, Yoshimi A, Shimabe M, et al. Evi-1 is a transcriptional
target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells. [26]
Ono
R,
Masuya
MLL-fusion-mediated
M AN U
Blood. 2011;117:6304-6314.
M,
Nakajima
leukemogenesis
H,
et
specifically
al.
Plzf
in
drives
long-term
hematopoietic stem cells. Blood. 2013;122:1271-1283. [27]
Zhang Y, Owens K, Hatem L, et al. Essential role of PR-domain
protein MDS1-EVI1 in MLL-AF9 leukemia. Blood. 2013;122:2888-2892. Placke T, Faber K, Nonami A, et al. Requirement for CDK6 in
TE D
[28]
MLL-rearranged acute myeloid leukemia. Blood. 2014;124:13-23. [29]
van der Linden MH, Willekes M, van Roon E, et al. MLL
fusion-driven
activation
of
CDK6
potentiates
proliferation
in
[30]
EP
MLL-rearranged infant ALL. Cell cycle. 2014;13:834-844. Laurenti E, Frelin C, Xie S, et al. CDK6 levels regulate quiescence
AC C
exit in human hematopoietic stem cells. Cell Stem Cell. 2015;16:302-313. [31]
Ayton PM, Cleary ML. Transformation of myeloid progenitors by
MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev. 2003;17:2298-2307. [32]
Milne TA, Briggs SD, Brock HW, et al. MLL targets SET domain
methyltransferase
activity
to
Hox
gene
promoters.
Molecular
cell.
2002;10:1107-1117. [33]
Nakamura T, Mori T, Tada S, et al. ALL-1 is a histone
methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Molecular cell. 2002;10:1119-1128. 23
ACCEPTED MANUSCRIPT
[34]
Yu BD, Hess JL, Horning SE, Brown GA, Korsmeyer SJ. Altered
Hox expression and segmental identity in Mll-mutant mice. Nature. 1995;378:505-508. [35]
Yagi H, Deguchi K, Aono A, Tani Y, Kishimoto T, Komori T. Growth
RI PT
disturbance in fetal liver hematopoiesis of Mll-mutant mice. Blood. 1998;92:108-117. [36]
Sedkov Y, Tillib S, Mizrokhi L, Mazo A. The bithorax complex is
regulated by trithorax earlier during Drosophila embryogenesis than is the
SC
Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early trithorax transcripts. Development. 1994;120:1907-1917. [37]
Breen TR, Harte PJ. Trithorax regulates multiple homeotic genes in
M AN U
the bithorax and Antennapedia complexes and exerts different tissue-specific, parasegment-specific and promoter-specific effects on each. Development. 1993;117:119-134. [38]
Deschamps J, van Nes J. Developmental regulation of the Hox
genes
during
axial
morphogenesis
2005;132:2931-2942.
the
mouse.
Development.
Wang KC, Helms JA, Chang HY. Regeneration, repair and
TE D
[39]
in
remembering identity: the three Rs of Hox gene expression. Trends in cell biology. 2009;19:268-275.
Yu BD, Hanson RD, Hess JL, Horning SE, Korsmeyer SJ. MLL, a
mammalian
trithorax-group
EP
[40]
gene,
functions
as
a
transcriptional
maintenance factor in morphogenesis. Proc Natl Acad Sci U S A.
AC C
1998;95:10632-10636. [41]
Blobel GA, Kadauke S, Wang E, et al. A reconfigured pattern of
MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit. Molecular cell. 2009;36:970-983. [42]
Jude CD, Climer L, Xu D, Artinger E, Fisher JK, Ernst P. Unique
and independent roles for MLL in adult hematopoietic stem cells and progenitors. Cell Stem Cell. 2007;1:324-337. [43]
Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM,
Weissman IL. Differential expression of novel potential regulators in hematopoietic stem cells. PLoS Genet. 2005;1:e28. 24
ACCEPTED MANUSCRIPT
[44]
Yokoyama A, Ficara F, Murphy MJ, et al. MLL becomes functional
through intra-molecular interaction not by proteolytic processing. PloS one. 2013;8:e73649. [45]
Somervaille TC, Cleary ML. Identification and characterization of
RI PT
leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 2006;10:257-268. [46]
Yokoyama A, Ficara F, Murphy MJ, et al. Proteolytically cleaved
MLL subunits are susceptible to distinct degradation pathways. J Cell Sci. [47]
SC
2011;124:2208-2219.
McMahon KA, Hiew SY, Hadjur S, et al. Mll has a critical role in
fetal and adult hematopoietic stem cell self-renewal. Cell Stem Cell. [48]
M AN U
2007;1:338-345.
Artinger EL, Ernst P. Cell context in the control of self-renewal and
proliferation regulated by MLL1. Cell cycle. 2013;12:2969-2972. [49]
Slany RK, Lavau C, Cleary ML. The oncogenic capacity of
HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol Cell Biol. 1998;18:122-129. Ayton PM, Chen EH, Cleary ML. Binding to nonmethylated CpG
TE D
[50]
DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein. Mol Cell Biol. 2004;24:10470-10478. [51]
Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O,
oncogenic
EP
Meyerson M, Cleary ML. The menin tumor suppressor protein is an essential cofactor
for
MLL-associated
leukemogenesis.
Cell.
AC C
2005;123:207-218. [52]
Yokoyama A, Cleary ML. Menin critically links MLL proteins with
LEDGF on cancer-associated target genes. Cancer Cell. 2008;14:36-46. [53]
Huang J, Gurung B, Wan B, et al. The same pocket in menin binds
both MLL and JUND but has opposite effects on transcription. Nature. 2012;482:542-546. [54]
Botbol Y, Raghavendra NK, Rahman S, Engelman A, Lavigne M.
Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro. Nucleic Acids Res. 2008. [55]
Okuda H, Kawaguchi M, Kanai A, et al. MLL fusion proteins link 25
ACCEPTED MANUSCRIPT
transcriptional coactivators to previously active CpG-rich promoters. Nucleic Acids Res. 2014;42:4241-4256. [56]
Eidahl JO, Crowe BL, North JA, et al. Structural basis for
high-affinity binding of LEDGF PWWP to mononucleosomes. Nucleic Acids [57]
RI PT
Res. 2013;41:3924-3936.
Zhu L, Li Q, Wong SH, et al. ASH1L Links Histone H3 Lysine 36
Dimethylation to MLL Leukemia. Cancer Discov. 2016;6:770-783. [58]
Kuo AJ, Cheung P, Chen K, et al. NSD2 links dimethylation of
SC
histone H3 at lysine 36 to oncogenic programming. Molecular cell. 2011;44:609-620. [59]
Barski A, Cuddapah S, Cui K, et al. High-resolution profiling of
[60]
M AN U
histone methylations in the human genome. Cell. 2007;129:823-837. Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K, Titgemeyer F,
Slany RK. The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation. Nucleic Acids Res. 2002;30:958-965. [61]
Allen MD, Grummitt CG, Hilcenko C, et al. Solution structure of the
TE D
nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase. Embo J. 2006;25:4503-4512. [62]
Cierpicki T, Risner LE, Grembecka J, et al. Structure of the MLL
CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. [63]
EP
Nat Struct Mol Biol. 2010;17:62-68. Milne TA, Kim J, Wang GG, et al. Multiple interactions recruit
AC C
MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis. Molecular cell. 2010;38:853-863. [64]
Muntean AG, Tan J, Sitwala K, et al. The PAF complex synergizes
with MLL fusion proteins at HOX loci to promote leukemogenesis. Cancer Cell. 2010;17:609-621. [65]
La P, Silva AC, Hou Z, et al. Direct binding of DNA by tumor
suppressor menin. J Biol Chem. 2004;279:49045-49054. [66]
Thiel
AT,
Blessington
P,
Zou
T,
et
al.
MLL-AF9-induced
leukemogenesis requires coexpression of the wild-type Mll allele. Cancer Cell. 2010;17:148-159. 26
ACCEPTED MANUSCRIPT
[67]
Mishra BP, Zaffuto KM, Artinger EL, et al. The histone
methyltransferase activity of MLL1 is dispensable for hematopoiesis and leukemogenesis. Cell reports. 2014;7:1239-1247. [68]
Garcia-Cuellar MP, Buttner C, Bartenhagen C, Dugas M, Slany RK.
RI PT
Leukemogenic MLL-ENL Fusions Induce Alternative Chromatin States to Drive a Functionally Dichotomous Group of Target Genes. Cell reports. 2016;15:310-322. [69]
Wang Z, Song J, Milne TA, et al. Pro isomerization in MLL1 cassette
connects
H3K4me
readout
to
CyP33
and
SC
PHD3-bromo
HDAC-mediated repression. Cell. 2010;141:1183-1194. [70]
Li BE, Gan T, Meyerson M, Rabbitts TH, Ernst P. Distinct pathways
M AN U
regulated by menin and by MLL1 in hematopoietic stem cells and developing B cells. Blood. 2013;122:2039-2046. [71]
Nilson I, Reichel M, Ennas MG, et al. Exon/intron structure of the
human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia. Br J Haematol. 1997;98:157-169.
Estable MC, Naghavi MH, Kato H, et al. MCEF, the newest member
TE D
[72]
of the AF4 family of transcription factors involved in leukemia, is a positive transcription
elongation
2002;9:234-245.
protein.
J
Biomed
Sci.
Benedikt A, Baltruschat S, Scholz B, et al. The leukemogenic
EP
[73]
factor-b-associated
AF4-MLL fusion protein causes P-TEFb kinase activation and altered
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epigenetic signatures. Leukemia. 2011;25:135-144. [74]
Lin C, Smith ER, Takahashi H, et al. AFF4, a component of the
ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Molecular cell. 2010;37:429-437. [75]
Bitoun E, Oliver PL, Davies KE. The mixed-lineage leukemia fusion
partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates
coordinated
chromatin
remodeling.
Hum
Mol
Genet.
2007;16:92-106. [76]
Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway JW.
An RNA polymerase II elongation factor encoded by the human ELL gene. 27
ACCEPTED MANUSCRIPT
Science. 1996;271:1873-1876. [77]
Peterlin BM, Price DH. Controlling the elongation phase of
transcription with P-TEFb. Molecular cell. 2006;23:297-305. [78]
Sobhian B, Laguette N, Yatim A, et al. HIV-1 Tat assembles a
the 7SK snRNP. Molecular cell. 2010;38:439-451. [79]
RI PT
multifunctional transcription elongation complex and stably associates with He N, Liu M, Hsu J, et al. HIV-1 Tat and host AFF4 recruit two
transcription elongation factors into a bifunctional complex for coordinated [80]
SC
activation of HIV-1 transcription. Molecular cell. 2010;38:428-438.
Mueller D, Bach C, Zeisig D, et al. A role for the MLL fusion partner
2007;110:4445-4454. [81]
M AN U
ENL in transcriptional elongation and chromatin modification. Blood. Li Y, Wen H, Xi Y, et al. AF9 YEATS domain links histone
acetylation to DOT1L-mediated H3K79 methylation. Cell. 2014;159:558-571. [82]
Okuda H, Kanai A, Ito S, Matsui H, Yokoyama A. AF4 uses the SL1
components
of
RNAP1
machinery
to
initiate
MLL
fusion-
and
AEP-dependent transcription. Nat Commun. 2015;6:8869. Mueller D, Garcia-Cuellar MP, Bach C, Buhl S, Maethner E, Slany
TE D
[83]
RK. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 2009;7:e1000249. [84]
Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a
EP
molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer. 2010;10:721-728.
Okuda H, Takahashi S, Takaori-Kondo A, Yokoyama A. TBP loading
AC C
[85]
by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription. Cell cycle. In press. [86]
Learned RM, Cordes S, Tjian R. Purification and characterization of
a transcription factor that confers promoter specificity to human RNA polymerase I. Mol Cell Biol. 1985;5:1358-1369. [87]
Comai L, Zomerdijk JC, Beckmann H, Zhou S, Admon A, Tjian R.
Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits. Science. 1994;266:1966-1972. [88]
Eberhard D, Tora L, Egly JM, Grummt I. A TBP-containing 28
ACCEPTED MANUSCRIPT
multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I. Nucleic Acids Res. 1993;21:4180-4186. of
Gorski JJ, Pathak S, Panov K, et al. A novel TBP-associated factor SL1
functions
in
RNA polymerase
I
transcription.
2007;26:1560-1568. [90]
Goodfellow
SJ,
Zomerdijk
JC.
Basic
EMBO
J.
RI PT
[89]
mechanisms
in
RNA
polymerase I transcription of the ribosomal RNA genes. Sub-cellular biochemistry. 2013;61:211-236.
Takahashi H, Parmely TJ, Sato S, et al. Human mediator subunit
SC
[91]
MED26 functions as a docking site for transcription elongation factors. Cell. 2011;146:92-104.
Malik S, Roeder RG. The metazoan Mediator co-activator complex
M AN U
[92]
as an integrative hub for transcriptional regulation. Nat Rev Genet. 2010;11:761-772. [93]
Kuntimaddi A, Achille NJ, Thorpe J, et al. Degree of Recruitment of
DOT1L to MLL-AF9 Defines Level of H3K79 Di- and Tri-methylation on Target Genes and Transformation Potential. Cell reports. 2015;11:808-820. Feng Q, Wang H, Ng HH, et al. Methylation of H3-lysine 79 is
TE D
[94]
mediated by a new family of HMTases without a SET domain. Curr Biol. 2002;12:1052-1058. [95]
Jones B, Su H, Bhat A, et al. The histone H3K79 methyltransferase
EP
Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet. 2008;4:e1000190. Chen
CW,
AC C
[96]
Koche
RP,
Sinha
AU,
et
al.
DOT1L inhibits
SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nat Med. 2015;21:335-343. [97]
Biswas D, Milne TA, Basrur V, et al. Function of leukemogenic
mixed lineage leukemia 1 (MLL) fusion proteins through distinct partner protein complexes. Proc Natl Acad Sci U S A. 2011;108:15751-15756. [98]
Bernt KM, Zhu N, Sinha AU, et al. MLL-rearranged leukemia is
dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell. 2011;20:66-78. [99]
Chang MJ, Wu H, Achille NJ, et al. Histone H3 lysine 79 29
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methyltransferase Dot1 is required for immortalization by MLL oncogenes. Cancer Res. 2010;70:10234-10242. [100]
Nguyen AT, Taranova O, He J, Zhang Y. DOT1L, the H3K79
Blood. 2011;117:6912-6922. [101]
RI PT
methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Jo SY, Granowicz EM, Maillard I, Thomas D, Hess JL. Requirement
for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation. Blood. 2011;117:4759-4768.
Daigle SR, Olhava EJ, Therkelsen CA, et al. Selective killing of
SC
[102]
mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011;20:53-65.
Daigle SR, Olhava EJ, Therkelsen CA, et al. Potent inhibition of
M AN U
[103]
DOT1L as treatment of MLL-fusion leukemia. Blood. 2013;122:1017-1025. [104]
Grembecka J, He S, Shi A, et al. Menin-MLL inhibitors reverse
oncogenic activity of MLL fusion proteins in leukemia. Nature chemical biology. 2012;8:277-284. [105]
Borkin D, He S, Miao H, et al. Pharmacologic Inhibition of the
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Menin-MLL Interaction Blocks Progression of MLL Leukemia In Vivo.
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Cancer Cell. 2015;27:589-602.
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Figure legends
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FIGURE 1 Models of gene activation by MLL and MLL fusion complexes. Expression of
self-renewal-related genes in MLL-r leukemia (A) and normal hematopoiesis (B). The cartoon
The mechanisms of target recognition by MLL and MLL fusion complexes.
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FIGURE 2.
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was modified from a previous review article [6]
(A) Schematic structures of MLL and MLL fusion complexes. The domain structures
responsible for various protein-protein interactions are indicated. hMBM: high affinity menin
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binding motif; LBD: LEDGF binding domain; PHD: plant homeodomain; HBM; HCF binding
motif; AD: activation domain; PS: processing site; Win: WDR5 interaction motif; IBD:
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Integrase binding domain; MTM: minimum targeting module. The cartoon was modified from a
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previous review article [6]. (B) Schematic representation of the mechanisms by which MLL and
MLL fusion complexes recognize their target chromatin.
FIGURE 3.
The domains/functions required for gene activation by MLL-AEP fusion
proteins. (A) The functions of various domains of AF4 are depicted schematically. The abilities
to transform myeloid progenitors and activate Hoxa9 expression in the form of MTM fusion are
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indicated along with various functions. The ALF and pSER domains were originally defined by
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Nilson et al. [71]. Their definition was slightly modified as the DLXLS motif, which was
originally included in the ALF domain, was included in the pSER domain in our reports [82, 85].
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(B) The functions of various subdomains in the pSER domain are depicted schematically.
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FIGURE 4. Model of gene activation by MLL-AEP fusion proteins. The rate-limiting step
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carried out by MLL-AEP fusion proteins is highlighted in red.
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Highlights
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• • •
MLL gene rearrangements cause acute leukemia by constitutive activation of self-renewal-related genes MLL fusion proteins target the promoter proximal regions of previously-active CpG-rich genes TBP loading through SL1 is the rate-limiting step regulated by MLL-AEP fusion proteins. Recruitment of transcription elongation and mediator activities by MLL fusion proteins is not essential for leukemogenesis.
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•