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HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis

  1. Satoshi Takahashi
  2. Akinori Kanai
  3. Hiroshi Okuda
  4. Ryo Miyamoto
  5. Yosuke Komata
  6. Takeshi Kawamura
  7. Hirotaka Matsui
  8. Toshiya Inaba
  9. Akifumi Takaori-Kondo
  10. Akihiko Yokoyama  Is a corresponding author
  1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Japan
  2. Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Japan
  3. Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
  4. Isotope Science Center, The University of Tokyo, Japan
  5. Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto University, Japan
  6. Division of Hematological Malignancy, National Cancer Center Research Institute, Japan
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Cite this article as: eLife 2021;10:e65872 doi: 10.7554/eLife.65872

Abstract

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

Introduction

Mutated transcriptional regulators often cause uncontrolled self-renewal of immature hematopoietic precursors, which leads to aggressive leukemia. MLL (also known as KMT2A) is a transcriptional maintenance factor that upregulates homeobox (HOX) genes in development (Yu et al., 1998). Chromosomal translocations generate fusion genes encoding various MLL fusion proteins with more than 80 different partners to induce leukemia (Meyer et al., 2018). MLL fusion proteins cause uncontrolled self-renewal by constitutively activating various oncogenic genes (e.g., HOXA9, MEIS1), whose expression is normally restricted in immature precursors such as hematopoietic stem cells (HSCs) (Krivtsov et al., 2006). However, the mechanisms by which MLL fusion proteins aberrantly activate the expression of HSC-specific genes remain elusive.

MLL fusion proteins form a complex with MENIN (Yokoyama et al., 2005; Yokoyama et al., 2004), which leads to further association with LEGDF (Yokoyama and Cleary, 2008). MLL fusion proteins bind to their target chromatin through the CXXC domain of MLL, which specifically recognizes unmethylated CpGs, and the PWWP domain of LEDGF, which selectively binds to di/tri-methylated histone H3 lysine 36 (H3K36me2/3) (Ayton et al., 2004; Birke et al., 2002; Pradeepa et al., 2012; van Nuland et al., 2013). The CXXC and PWWP domains constitute the minimum targeting module (MTM) which can stably bind to the human MLL target gene promoters (e.g., HOXA9) (Okuda et al., 2014). Because unmethylated CpGs and H3K36me2/3 marks are associated with transcriptional activation, MLL fusion proteins target a broad range of previously transcribed CpG-rich promoters. Although MLL fuses with a variety of partners, most MLL fusion-mediated leukemia cases are caused by the fusions with the AF4 family (e.g., AF4 also known as AFF1, AF5Q31 also known as AFF4) and ENL family (e.g., ENL also known as MLLT1, AF9 also known as MLLT3) (Meyer et al., 2018). AF4 family proteins form a biochemically stable complex with ENL family proteins and the P-TEFb elongation factor (composed of CDK9 and CyclinT1/2), which we termed AEP complex (as in AF4 family/ENL family/P-TEFb complex) (Yokoyama et al., 2010). AF4 family proteins recruit the SL1 complex (Okuda et al., 2015), which is composed of TBP and the TAF1A/B/C/D subunits and is known to initiate ribosomal RNA transcription by RNA polymerase I (Goodfellow and Zomerdijk, 2013). An artificial construct of MTM fused to the binding platform for SL1 activated the murine Hoxa9 gene and transformed hematopoietic progenitors (HPCs) (Okuda et al., 2015), indicating that the AF4 family protein activates RNA polymerase II (RNAP2)-dependent transcription, presumably by loading TBP onto the target promoters via SL1. However, why MLL fusion proteins preferentially use the AEP/SL1-mediated transcription pathway is unclear.

In this study, we identified the evolutionarily conserved trithorax homology domain 2 (THD2) of MLL (previously termed TRX2 domain) as a key structure required for aberrant self-renewal mediated by MLL fusion proteins. A subsequent proteomic approach identified the HBO1 (also known as KAT7 and MYST2) histone acetyltransferase (HAT) complex as an associating factor for THD2. HBO1 is a member of the MYST HAT family responsible for histone H3 lysine 14 acetylation (H3K14ac) when complexed with BRPF family proteins, while it mainly acetylates histone H4 lysine 5/8/12 when complexed with JADE family proteins (Lalonde et al., 2013; Mishima et al., 2011). HBO1 was recently identified as a therapeutic vulnerability of leukemia stem cells by genetic screening (Au et al., 2020; MacPherson et al., 2020). However, its molecular functions on leukemic proteins remain largely elusive. Our detailed structure/function analysis demonstrated that HBO1-MLL interaction promotes AEP-dependent gene activation in MLL fusion-mediated leukemic transformation. Moreover, another leukemic fusion of nucleoporin-98 (NUP98) and HBO1(i.e., NUP98-HBO1) also transformed HPCs via association with MLL. Hence, we propose that HBO1-MLL interaction modules can be utilized as molecular targets for developing drugs that specifically dismantle the oncogenic transcriptional machinery.

Results

THD2-mediated functions promote MLL fusion-dependent leukemic transformation

MLL fusion proteins confer unlimited self-renewal ability, leading to the immortalization of HPCs (Lavau et al., 1997). MLL-ELL is among the frequently observed MLL fusions associated with acute myelogenous leukemia (Meyer et al., 2018). To determine the domain structures required for MLL-ELL-mediated leukemic transformation, we performed myeloid progenitor transformation assays (Lavau et al., 1997; Okuda and Yokoyama, 2017b), wherein murine HPCs were retrovirally transduced with an MLL fusion gene and cultured in a semisolid medium supplemented with myeloid cytokines (Figure 1A). MLL-ELL transformed HPCs, as previously reported (DiMartino et al., 2000; Luo et al., 2001), featured vigorous colony-forming capacities at the third and fourth passages, and showed elevated Hoxa9 expression at the first and second passages. A deletion mutant lacking THD2 failed to transform (Figure 1A and Figure 1—figure supplement 1A), underscoring the biological significance of THD2. A minimalistic artificial construct of HA-tagged MTM fused to an intact Occludin homology domain (OHD) of ELL failed to transform HPCs (Figure 1A, see MTMh-ELL″), whereas inclusion of THD2 to MTM (hereafter denoted as MTMT) caused constitutive activation of Hoxa9 and immortalization of HPCs (Figure 1A, see MTMTh-ELL″). These results indicate that MLL-ELL transforms HPCs through THD2-mediated functions.

Figure 1 with 1 supplement see all
Trithorax homology domain 2 (THD2)-mediated functions promote MLL-fusion-dependent leukemic transformation.

(A) Structure/function analysis of MLL-ELL. Various MLL-ELL constructs were examined for the transformation of myeloid progenitors. HA-tag (h: indicated as a red triangle) was fused to MTM and MTMT constructs. A schema of myeloid progenitor transformation assay is shown on top. Hoxa9 expression normalized to Gapdh in first-round and second-round colonies (left) is shown as the relative value of MLL-ELL (arbitrarily set at 100%) with error bars (mean ± SD of PCR triplicates). Colony-forming ability at third- and fourth-round passages (right) is shown with error bars (mean ± SD of ≥3 biological replicates). IBD: integrase-binding domain; hMBM: high-affinity MENIN-binding motif; LBD: LEDGF-binding domain; THD1/2: trithorax homology domains 1 and 2; SET: Suver3-9/enhancer-of-zeste/trithorax motif; TED: transcription elongation domain; OHD: occludin homology domain. (B) Requirement of THD2 for various MLL fusion proteins in leukemic transformation. Various MLL fusion constructs were examined for the transformation of myeloid progenitors as in (A). RA1/2: RAS association domains 1/2; PDZ: PSD-95/Dlg/ZO-1 domain; OM: octapeptide motif; LZ: leucine zipper. MISD: minimum interaction site of DOT1L: YEATS: Yaf9 ENL AF9 Taf14 Sas5 domain; AHD: ANC1 homology domain. (C) Colony morphologies of MTMTh or MTMh-ENL′ transformed cells. The colonies on day 5 of fourth passage are shown with a scale bar. The ratio of compact colonies (≥100 total colonies were counted in each experiment) is shown on the left (mean ± SD of six biological replicates). Representative images are shown on the right.

Next, we tested the structural requirements of other MLL fusions (i.e., MLL-AF6, MLL-AF10, and MLL-ENL) (Figure 1B and Figure 1—figure supplement 1A). An MLL-AF6 fusion construct, in which MLL is fused to the RA1 and RA2 domains of AF6 (see MLL-AF6′), fully transformed HPCs as previously reported (Liedtke et al., 2010). The minimalistic MTM-AF6 fusion construct (Figure 1B, see MTMh-AF6′) activated Hoxa9 expression in the early passages but produced modest numbers of colonies in later passages, while the inclusion of THD2 (Figure 1B, see MTMTh-AF6′) conferred much more vigorous transforming capacities. Deletion of THD2 from a PWWP-MLL-AF6 fusion construct containing the residues 641/1267 of MLL (P′641/1267h-AF6′) abrogated its transforming ability, suggesting that MLL-AF6 requires THD2 to exert its full transforming potential (Figure 1B and Figure 1—figure supplement 1A). Nonetheless, an MLL-AF6′ construct lacking THD2 immortalized HPCs albeit less efficiently compared to MLL-AF6′ (Figure 1—figure supplement 1B, see MLL-AF6′ ΔTHD2), suggestive of some compensatory functions mediated by other MLL structures retained in MLL-AF6′ but missing in P′641/1267h-AF6′ (i.e., the residues 1/640 and 1268/1395). MLL-AF10 showed a similar trend, in line with our previous report (Okuda et al., 2017Figure 1B and Figure 1—figure supplement 1A,B). In contrast, the oncogenic properties of MLL-ENL were not severely affected by the loss of THD2 in terms of colony-forming capacity (Figure 1B and Figure 1—figure supplement 1BOkuda et al., 2014). However, the colony morphology of immortalized cells (ICs) transformed by the MTMh-ENL construct (MTMh-ENL′-ICs) was more differentiated compared to that of MTMTh-ENL′-ICs (Figure 1C), suggesting that THD2 is required to block differentiation to some extent. Leukemogenesis in vivo was compromised by the deletion of THD2 for MLL-AF6, -AF10, and -ENL (Figure 1—figure supplement 1C). These results indicate that MLL fusion proteins rely on THD2-mediated functions for leukemic transformation to varying degrees depending on their fusion partners. Among the MLL fusions tested, MLL-ELL most heavily relies on THD2-mediated functions.

HBO1 complex associates with MLL proteins via THD2 at promoters

We previously showed that THD2 binds to AF4 family proteins (Okuda et al., 2017). Indeed, a FLAG-tagged MLL construct encompassing the residues 869/1152 efficiently co-precipitated with exogenously expressed AF4 or AF5Q31 (Figure 2—figure supplement 1A, see fMLL 869/1152+37aa). However, deletion of THD2 from the FLAG-tagged MLLΔFP construct (containing the residues 1/1395: fMLL ΔFP) did not impair co-precipitation of AF4, indicating that the interaction with AF4 is not mediated by THD2. Sequencing analysis of the vector constructs revealed that the fMLL 869/1152+37aa construct contained an additional coding sequence tethered in frame for 37 residues derived from the pCMV5 vector, which corresponds to a part of the chorionic somatomammotropin hormone 1 gene (Figure 2—figure supplement 1B). Removal of the additional 37 residues by introducing a stop codon resulted in complete loss of association with AF4 family proteins (Figure 2—figure supplement 1A, see fMLL 869/1251). Moreover, a FLAG-tagged GAL4 fusion construct tethered to the additional 37 residues co-precipitated with AF5Q31 (see fGAL4+37aa). Thus, we concluded that our previous claim for THD2 as a binding platform for AF4 family proteins was false.

To identify bona fide associating factors for THD2, we purified two exogenously expressed THD2-containing proteins (i.e., fGAL4-MLL 869/1124 and MTMTh) from the chromatin fraction of HEK293T cells using the fractionation-assisted chromatin immunoprecipitation (fanChIP) method (Miyamoto and Yokoyama, 2021) and analyzed by mass spectrometry (Figure 2A). Components of the HBO1 complex including HBO1, PHF16 (also known as JADE3), MEAF6, and ING5 were detected in the purified materials (Avvakumov et al., 2012). Immunoprecipitation-western blotting (IP-WB) analysis confirmed that fGAL4-MLL 869/1124 co-precipitated with HBO1 complex components but not with exogenously expressed AF4 (Figure 2B). A series of MTMT fusion proteins were associated with HBO1 and PHF16, while the respective MTM fusion proteins were not (Figure 2C), confirming that THD2 mediates interaction with the HBO1 complex. Domain mapping analysis of MLL showed that the residues 869/1124 contain the major binding domains for the HBO1 complex (Figure 2—figure supplement 2A). Moreover, fGAL4-MLL 1052/1124 co-precipitated with the HBO1 complex components after DNaseI treatment, indicating that association of MLL and HBO1 is not mediated by DNA (Figure 2—figure supplement 2B). It should be noted that a small amount of HBO1 co-precipitated with the MLL proteins lacking the THD2 domain (Figure 2—figure supplement 2C, see fMLL ΔFP ΔTHD2 and fMLL-ENL ΔTHD2), suggesting that there is a secondary binding domain for the HBO1 complex outside of THD2, which may account for the moderate effects of THD2 deletion from the full-length MLL fusion constructs (Figure 1—figure supplement 1B).

Figure 2 with 3 supplements see all
HBO1 complex associates with MLL proteins at promoters.

(A) Purification of trithorax homology domain 2 (THD2) domain-associating factors. A FLAG-tagged (f: indicated as a blue triangle) GAL4 DNA-binding domain fused with the MLL fragment containing the residues 869–1124 or HA-tagged MTMT fragment was transiently expressed in HEK293T cells. A schema of the fractionation-assisted chromatin immunoprecipitation (fanChIP) method is shown on top. The transgene products were purified from the chromatin fraction and analyzed by mass spectrometry. Silver-stained images (right) of the purified materials are shown. Asterisk indicates the position of the transgene products. A Venn diagram of identified THD2 domain-associating factors by mass spectrometry is shown. (B) Association of GAL4-THD2 fusion with HBO1. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293T cells transiently expressing FLAG-tagged GAL4-MLL 869/1124 construct and Xpress-tagged AF4 (xAF4) was performed. Co-purification of HBO1, but not xAF4, was confirmed. #: the sample used for the input. (C) THD2-dependent association with the HBO1 complex. IP-western blotting of the chromatin fraction of virus-packaging cells, transiently expressing various HA-tagged MTMT (or MTM) fusion constructs, was performed. (D) Genomic localization of MLL and the HBO1 complex components in HB1119 cells. The ChIP followed by deep sequencing (ChIP-seq) profiles were visualized using the Integrative Genomics Viewer (The Broad Institute). The minimum value of the y-axis was set at 0, while the maximum value for each sample is indicated. (E) Average distribution of proteins near the transcription start sites (TSSs) of HB1119 cells. Genes whose MLL ChIP signal/input ratio at the promoter proximal transcribed region was ≥5 were defined as MLL target genes. Average ChIP signal distribution of indicated proteins at the MLL target genes (red) or all genes (black) is shown. The y-axis indicates the frequency of the ChIP-seq tag count (ppm) in 25 bp increments. (F) Requirement of HBO1 for myeloid progenitors immortalized by various oncogenes. sgRNA competition assays for Hbo1 were performed on immortalized myeloid progenitors. The ratio of GFP-positive cells co-expressing sgRNA was measured by flow cytometry. sgRNA for Renilla luciferase (Ren) was used as a negative control, which does not affect proliferation. sgRNA for Rpa3 was used as a positive control, which impairs proliferation.

Interaction between endogenous MLL proteins and HBO1 was confirmed in leukemia cell lines such as HB1119 and REH (Figure 2—figure supplement 2D,E). Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) of HB1119 cells, which endogenously express MLL-ENL, demonstrated that HBO1 complex components colocalized with MLL-ENL at the MLL target genes (e.g., MYC, HOXA9, and MYB) in a genome-wide manner (Figure 2D,E and Figure 2—figure supplement 3A). CRISPR/Cas9-mediated sgRNA competition assays demonstrated that Kat7/KAT7 (the gene encoding HBO1) is required for the proliferation of various MLL fusion-ICs (Figure 2F) and in human leukemia cell lines (Figure 2—figure supplement 3B). These results are consistent with the recent reports, which showed that the HBO1 complex associates with multiple MLL fusion proteins and plays a critical role in the maintenance of leukemia stem cells (Au et al., 2020; MacPherson et al., 2020). Taken together, MLL fusion proteins associate with the HBO1 complex through THD2 at the target promoters.

MLL recruits the HBO1, AEP, and SL1 complexes to promoters

To elucidate the functional relationship between wild-type MLL and the HBO1 complex, we examined the genomic localization of the HBO1 complex, wild-type MLL, and its related transcriptional regulators in HEK293T cells (Figure 3A and Figure 3—figure supplement 1A). MLL localizes at the transcription start sites (TSSs) of CpG-rich genes including RPL13A, MYC, and CDKN2C (Okuda et al., 2017). Distribution of the HBO1 complex was enriched at promoter proximal transcribed regions (0–2 kb from the TSS) (Figure 3B) consistent with a previous report (Avvakumov et al., 2012), suggesting its implication in transcription initiation/elongation. The HBO1 complex was localized at the MLL-occupied promoters in a genome-wide manner similarly to that in HB1119 cells. We previously showed that MLL associates with the MOZ complex and yet-to-be activated RNAP2 whose heptapeptide repeats are not phosphorylated (RNAP2 non-P) on target promoters, and that it colocalizes with various ENL-containing complexes including the AEP and DOT1L complexes (Miyamoto et al., 2020; Okuda et al., 2017). The ChIP signal intensities of MLL were highly correlated with those of HBO1, MOZ, and RNAP2 non-P (Figure 3C), supporting the functional interactions of MLL with those MLL-associated factors. The ChIP signals of AEP (e.g., AF4, ENL) and SL1 (e.g., TAF1C) are weakly correlated with that of MLL, presumably because AEP and SL1 are indirectly recruited to MLL target promoters in a context-dependent manner.

Figure 3 with 1 supplement see all
MLL recruits the HBO1 and AEP/SL1 complexes to promoters.

(A) Genomic localization of various transcriptional regulators/epigenetic marks in HEK293T cells. Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analysis was performed on the chromatin of HEK293T cells for the indicated proteins/modifications. CIRA-seq data for unmethylated CpGs (unMe CpGs) and RNA-seq data are shown for comparison. (B) Average distribution of proteins near transcription start sites (TSSs) of HEK293T cells. Average ChIP signal distribution of indicated proteins at the MLL target genes (red) or all genes (black) is shown as in Figure 2E. (C) Relative occupation by multiple factors at all TSSs. ChIP-seq or CIRA-seq tags of indicated proteins at all genes were clustered into a 2 kb bin (0 to +2 kb from the TSS) and are presented as XY scatter plots. Spearman’s rank correlation coefficient (ρ) is shown. (D) Expression of MLL in MLL-deficient HEK293T cell lines. (E) Expression of MLL target genes in two independently generated MLL-deficient clones. Relative expression levels normalized to GAPDH are shown with error bars (mean ± SD of PCR triplicates) by RT-qPCR. Data are redundant with our previous report (Miyamoto et al., 2020). (F) Localization of MLL, the HBO1 complex, the AEP complex, and the SL1 complex in two independently generated MLL-deficient clones. ChIP-qPCR was performed for indicated genes using qPCR probes designed for the TSS of each gene. ChIP signals are expressed as the percent input with error bars (mean ± SD of biological triplicates). Statistical analysis was performed by ordinary two-way ANOVA comparing each sample with the parent cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

To examine the role of MLL in the genomic localization of the HBO1 complex, we analyzed MLL-deficient HEK293T cells by qRT-PCR and ChIP-qPCR. Depletion of MLL by the knockout of KMT2A (the gene encoding MLL) reduced the expression of MYC and CDKN2C (Figure 3D,E), as previously reported (Miyamoto et al., 2020), and caused a marked reduction in the ChIP signals of the HBO1 complex components (i.e., PHF16, MEAF6) at the MYC and CDKN2C loci (Figure 3F and Figure 3—figure supplement 1B). The HBO1 complex is presumed to target the chromatin containing histone H3 lysine 4 (H3K4me3) marks to which the PHD finger of the ING5 subunit specifically binds (Champagne et al., 2008). H3K4me3 marks were substantially reduced at the MYC and CDKN2C promoters as they were presumably provided by MLL in part. These results suggest that the decreased presence of the HBO1 complex may be due to the reduced H3K4me3 marks. Transient expression of a THD2-containing MLL construct (MTMTh) did not rescue the recruitment of the HBO1 complex onto the MYC promoter (Figure 3—figure supplement 1C), suggesting that H3K4 methylation is a prerequisite for the recruitment of the HBO1 complex. In addition, the ChIP signals of AEP/SL1 components (i.e., ENL, CCNT1, AF4, and TAF1C) were also reduced at these loci (Figure 3F and Figure 3—figure supplement 1B). These results indicate that MLL recruits the HBO1, AEP, and SL1 complexes to the MYC and CDKN2C promoters in part via H3K4 methylation.

MLL-ELL transforms through the common binding platform for AF4 and EAF1

Because MLL-ELL is highly dependent on THD2-mediated HBO interaction, we next dissected the function of the ELL portion in MLL-ELL-mediated leukemic transformation. ELL has a transcriptional elongation activity and is associated with a variety of proteins including AF4 and EAF family proteins (Lin et al., 2010; Shilatifard et al., 1996; Simone et al., 2003; Simone et al., 2001). Myeloid progenitor transformation assays demonstrated that an intact OHD, which is responsible for the association with both AF4 and EAF1 (Lin et al., 2010; Simone et al., 2001), is required for transformation as previously reported (Figure 4A,B and Figure 4—figure supplement 1ADiMartino et al., 2000; Luo et al., 2001). ChIP-qPCR analysis of FLAG-tagged GAL4 constructs fused to ELL confirmed that OHD is responsible for the recruitment of both AF4 and EAF1 (Figure 4—figure supplement 1B). Deletion of OHD resulted in the loss of interaction with both AF4 and EAF1 family proteins, which is correlated with the transforming properties (Figure 4B and Figure 4—figure supplement 1C). It should be noted that several processed forms of AF4 (e.g., 110 kDa) were observed in the co-precipitates, whose amounts were specifically increased in the chromatin fraction of HEK293T cells transiently expressing GAL4-ELL proteins harboring an intact OHD (Figure 4B and Figure 4—figure supplement 1C), suggesting that ELL induces the processing of AF4 and tethers both processed and unprocessed forms of AF4 to the chromatin. ELL-AF4 interaction was attenuated by co-expression of EAF1, whereas ELL-EAF1 interaction was augmented by co-expression of AF4 (Figure 4C,D), suggesting that these interactions occur sequentially but not simultaneously, wherein the ELL-AF4 interaction precedes the ELL-EAF1 interaction (Figure 4E). Taken together, MLL-ELL exerts its transforming properties through the common binding platform for AF4 and EAF1.

Figure 4 with 1 supplement see all
MLL-ELL transforms through the common binding platform for AF4 and EAF1.

(A) Requirement of the occludin homology domain (OHD) in MLL-ELL-mediated transformation. Various MLL-ELL constructs carrying mutations in the ELL portion were examined for the transformation of myeloid progenitors, as in Figure 1A. NHD: N-terminal homology domain; ALF: AF4/LAF4/FMR2 homology domain; pSER: poly-serine; A9ID: AF9 interaction domain; CHD: C-terminal homology domain; EHD: EAF family homology domain; DLXLS: DLXLS motif; SDE: SDE motif. (B) OHD-dependent association with AF4. Immunoprecipitation (IP)-western blotting of the chromatin fraction of virus-packaging cells, transiently expressing various HA-tagged MTMT-ELL fusion constructs, was performed. Endogenous AF4 proteins co-purified with MTMTh-ELL proteins were visualized by an anti-AF4 antibody. (C) Sequential recruitment of AF4 and EAF1 by ELL. HEK293TL cells (Okuda et al., 2015), which harbor GAL4-responsive reporter, were transfected with various combinations of FLAG-tagged GAL4 fusion proteins, xAF4, and HA-tagged EAF1 (EAF1h), and were subjected to chromatin immunoprecipitation (ChIP)-qPCR analysis. A qPCR probe near the GAL4-responsive elements (UAS) was used. The ChIP signals were expressed as the percent input with error bars (mean ± SD of PCR triplicates). TATA: TATA box; LUC: luciferase. (D) Effects of overexpression of AF4 or EAF1 on ELL complex formation. IP-western blotting of the chromatin fraction of HEK293TL cells, transiently expressing various combinations of FLAG-tagged GAL4-ELL proteins, xAF4, and EAF1h, was performed. (E) A model of the sequential association between ELL, AF4 family proteins, and EAF family proteins.

AF4 and EAF1 form two distinct SL1/MED26-containing complexes

AF4 and EAF1 family proteins share some structural similarities; both have the SDE motif enriched with serine, aspartic acid, and glutamic acid, and the DLXLS motif whose consensus sequence is LXXDLXLS (Figure 5A,B). The NKW motif was found in the AF4 family but not in the EAF family. The SDE motif of AF4 associates with the SL1 complex and its DLXLS motif associates with MED26 (Okuda et al., 2015; Okuda et al., 2016; Takahashi et al., 2011). IP-WB analysis demonstrated that EAF1 was also associated with SL1 and MED26 through its C-terminal domain containing the SDE and DLXLS motifs (Figure 5C, see fGAL4-EAF1-C). ChIP-qPCR analysis confirmed that the C-terminal portion of EAF1 recruited TAF1C and MED26 to the GAL4-responsive promoter (Figure 5D, see fGAL4-EAF1-C). Nevertheless, the GAL4-ELL fusion failed to associate with or recruit TAF1C and MED26 (Figure 5C,D, see fGAL4-ELL′), indicating that ELL-bound EAF1 or AF4 is unable to interact with TAF1C and MED26, and therefore must dissociate from ELL to form a complex with SL1 and MED26 (Figure 5E,F). It should be noted that GAL4-EAF1 failed to pull down endogenous AF4 and ENL (Figure 5C,D, see fGAL4-EAF1), while a GAL4-AF4 protein containing an ELL-binding domain (ALF) also failed to pull down exogenously expressed EAF1 (Figure 5C,D, see fGAL4-AF4-2N and C), indicating that the AF4/ELL/EAF1 trimer complex is unstable and that ELL mostly binds AF4 or EAF family proteins in a mutually exclusive manner.

Figure 5 with 1 supplement see all
AF4 and EAF1 form distinct SL1/MED26-containing complexes.

(A) A schema of the structures of AF4, ELL, and EAF1. ALF is responsible for interaction with ELL, pSER is responsible for interaction with SL1, and DLXLS is responsible for interaction with MED26. EAF family homology domain (EHD) is responsible for homodimerization and interaction with ELL. NKW:NKW motif responsible for transcriptional activation. (B) Alignment of the amino acid sequences of the SDE, DLXLS, and NKW motifs of human AF4 family proteins and EAF family proteins. Conserved residues are indicated by asterisks. (C) Association of EAF1, ELL, and AF4 domains with various associating factors on chromatin. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293TL cells transiently expressing various FLAG-tagged GAL4 fusion proteins, with or without indicated HA-tagged constructs, was performed as in Figure 4D. Endogenous proteins were detected by specific antibodies for each protein, while exogenous proteins were detected by antibodies for FLAG or HA tag. (D) Recruitment of various transcriptional regulatory proteins by EAF1, ELL, and AF4 domains. Chromatin immunoprecipitation (ChIP)-qPCR analysis of HEK293TL cells transiently expressing various combinations of FLAG-tagged GAL4 fusion proteins along with indicated HA-tagged constructs was performed as in Figure 4C. (E) Putative complex recruitment mediated by ELL-AF4 interaction. (F) Putative complex recruitment mediated by ELL-EAF1 interaction. (G) Structural requirement of MTM-AF4 pSER fusion and MTMT-EAF1 fusion. Various MTMh/MTMTh constructs fused with EAF1 domains were examined for the transformation of myeloid progenitors as in Figure 1A.

In addition, GAL4-EAF1 co-precipitated with exogenously expressed EAF1 through the EAF family homology domain (EHD), indicating that EAF1 forms a homodimer (Figure 5C, see the HA blot of exogenously expressed HA-tagged EAF1). It has been shown that EAF1 binds to ELL through two distinct contacts (Simone et al., 2003). Accordingly, both N- and C-terminal halves of EAF1 co-precipitated with endogenous ELL (Figure 5C, see fGAL4-EAF1-N and -C in the upper ELL blot). However, when EAF1 was overexpressed, co-precipitation of endogenous ELL by the C-terminal half of EAF1 was no longer detected (Figure 5C, see fGAL4-EAF1-C in the lower ELL blot), suggesting that free ELL preferentially binds to the EAF1 dimer over the monomeric C-terminal half of EAF1. GAL4-ELL co-precipitated with endogenous ELL presumably mediated by an EAF1 dimer (Figure 5C, see fGAL4-ELL′ in the upper ELL blot), whereas it failed to pull down ELL when exogenous ELL was overexpressed to absorb free EAF1 dimers (Figure 5C, see fGAL4-ELL′ in the HA blot of exogenously expressed HA-tagged ELL). Taken together, these results suggest that ELL forms at least two different stable complexes, one is with AF4 family proteins, which subsequently leads to the formation of an AEP/SL1/MED26 complex (Figure 5E), and the other is an EAF1/ELL homodimer complex, which leads to the formation of an EAF1/SL1/MED26 complex (Figure 5F). These two SL1/MED26 containing complexes are similar in composition but different in three key functions. The AEP/SL1/MED26 complex has the NKW motif which is required for AF4-dependent gene activation (Okuda et al., 2015), and contains P-TEFb which promotes transcription elongation, and ENL family proteins which tether AEP on acetylated chromatin (Erb et al., 2017; Li et al., 2014; Wan et al., 2017). Thus, we presumed that the AEP/SL1/MED26 complex is competent for transactivation whereas the EAF1/SL1/MED26 complex is not.

MLL-ELL-mediated leukemic transformation is driven by interaction with AEP, while MLL-EAF1-mediated transformation is promoted by homodimerization

Previously, Luo et al., 2001, demonstrated that an artificial fusion construct of MLL and EAF1 transformed HPCs and induced leukemia, which suggested that the ELL-EAF1 interaction, rather than ELL-AEP interaction, played a major role in MLL-ELL-mediated leukemic transformation. To examine the structural requirements for MLL-EAF1-mediated transformation, we generated artificial fusion constructs in which MTM or MTMT is fused to EAF1 domains and examined their transforming properties (Figure 5G and Figure 5—figure supplement 1A). As reported previously (Okuda et al., 2015), an MTM construct fused to an AF4 portion containing the SDE and NKW motifs (i.e., MTMh-AF4-2C-abc) activated Hoxa9 expression and immortalized HPCs, while a similar construct lacking the NKW motif (i.e., MTMh-AF4-2C-ab) failed to transform (Figure 5G). Accordingly, an MTM construct fused to the C-terminal half of EAF1 containing the SDE motif but lacking the NKW motif failed to transform HPCs (Figure 5G, see MTMh-EAF1-C). However, an MTM construct fused to the entire EAF1 demonstrated partial transforming properties, which maintained the expression of Hoxa9 in the early passages, but failed to immortalize HPCs (Figure 5G, see MTMh-EAF1). MTMT constructs fused to the EHD fully transformed HPCs (Figure 5G, see MTMTh-EAF1 and MTMTh-EAF1-N). This trend is reminiscent of MLL-AF6 which transforms HPCs by homodimerization (Figure 1BLiedtke et al., 2010) and suggests that MLL-EAF1 transforms by EHD-mediated homodimerization rather than by ELL-EAF1 interaction. Knockdown of Mllt1 (the gene encoding ENL) in MLL-ELL-ICs perturbed colony formation and Hoxa9 expression, while its effects on MLL-ENL-ICs were relatively limited presumably because MLL-ENL can directly recruit AEP (Figure 5—figure supplement 1B). With these results, we speculated that MLL-ELL transforms HPCs through the recruitment of AEP, while MLL-EAF1 transforms via homodimerization in a mechanism similar to MLL-AF6.

MLL-ELL transforms HPCs via association with AEP, but not with EAF1 or p53

To further evaluate the significance of ELL-AF4 and ELL-EAF1 interactions in MLL-ELL-mediated leukemic transformation, we introduced S600A/K606T double mutation (hereafter denoted as SA/KT) to the ELL portion, which was initially predicted to impair ELL-AF4 interaction based on the structural data for the ELL2/AF5Q31 complex (Qi et al., 2017). Indeed, the mutation severely attenuated ELL-AF4 interaction (Figure 6A, see fGAL4-ELL″ SA/KT). However, a substantial amount of AF4 (mostly the processed forms of AF4) remained associated with the SA/KT mutant, while co-precipitation of EAF1 was completely abolished. These results suggest that SA/KT mutation abolishes the primary contact of ELL for AF4 and EAF1, while there is an additional contact for AF4 that is unaffected by this mutation. Western blotting of the input samples of the chromatin fraction showed that the SA/KT mutant increased the amount of processed forms of AF4 in the chromatin fraction like the intact form (Figure 6A, see fGAL4-ELL″ and fGAL4-ELL″SA/KT in the Xpress blot), thus further confirming the interaction with fGAL4-ELL″ SA/KT and AF4. ChIP-qPCR analysis confirmed that exogenously expressed AF4 was recruited to the target chromatin by fGAL4-ELL″ SA/KT, while EAF1 was not (Figure 6B). Moreover, fGAL4-ELL″ SA/KT co-precipitated endogenous AF4 and AF5Q31 (Figure 6—figure supplement 1A), while it failed to pull down p53, another ELL associating factor (Wiederschain et al., 2003). fGAL4-ELL″ SA/KT recruited a substantial amount of endogenous ENL and CyclinT1 to the GAL4-responsive promoter (Figure 6—figure supplement 1B), indicating that the SA/KT mutant is competent for loading AEP onto chromatin.

Figure 6 with 1 supplement see all
MLL-ELL transforms hematopoietic progenitors via association with AF4/ENL/P-TEFb (AEP), but not with EAF1 or p53.

(A) Mutations of ELL selectively abrogate interaction with EAF1. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without S600A/K606T substitutions (SA/KT) along with xAF4 and EAF1h was performed as in Figure 4D. (B) Recruitment of exogenously expressed AF4 or EAF1 by ELL mutant proteins. Chromatin immunoprecipitation (ChIP)-qPCR analysis of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without the SA/KT mutation along with xAF4 and EAF1h was performed as in Figure 4C. (C) Effects of the SA/KT mutation on MLL-ELL-mediated leukemic transformation ex vivo. Various MLL-ELL constructs with or without the SA/KT mutation were examined for the transformation of myeloid progenitors as in Figure 1A. (D) Effects of the SA/KT mutation on MLL-ELL-mediated leukemic transformation in vivo. MLL-ELL or its SA/KT mutant was transduced to c-Kit-positive hematopoietic progenitors and transplanted into syngeneic mice (n = 20). Primary leukemia cells were harvested from the bone marrow and transplanted into secondary recipient mice (n = 20). (E) Enhanced interaction of ELL with AEP mediated by the trithorax homology domain 2 (THD2) domain. IP-western blotting of the chromatin fraction of virus-packaging cells, transiently expressing various HA-tagged MTMT-ELL fusion constructs (with or without the SA/KT mutation), was performed. Endogenous proteins co-purified with MLL-ELL proteins were visualized by specific antibodies.

Next, we examined the effects of the SA/KT mutation on the transforming properties of MLL-ELL. Both Hoxa9 expression and colony-forming potentials were maintained by MLL-ELL SA/KT-ICs (Figure 6C and Figure 6—figure supplement 1C). Remarkably, the MTM construct fused to ELL carrying the SA/KT mutation activated Hoxa9 and immortalized HPCs albeit with low clonogenicity despite the lack of THD2 (Figure 6C, see MTMh-ELL″ SA/KT), suggesting that the SA/KT mutation partially compensates for the lack of interaction with the HBO1 complex. MLL-ELL SA/KT induced leukemia in vivo (Figure 6D), indicating that direct recruitment of EAF1 or p53 is dispensable and potentially suppressive for MLL-ELL-mediated leukemic transformation. sgRNA competition assays showed that loss of Eaf1 has a marginal inhibitory effect on proliferation of MLL-ELL-ICs ex vivo, while loss of Trp53 accelerated it, suggestive of non-essential roles for EAF1 and an inhibitory role for p53 in MLL-ELL-mediated transformation (Figure 6—figure supplement 1D). IP-WB analysis of the MTMh- or MTMTh-ELL″ SA/KT showed that association with AF4 and ENL through the presumed secondary contact is enhanced by the presence of THD2 while p53 association remained abolished (Figure 6E). Leukemia cells of MLL-ELL were particularly sensitive to WM1119, a pan MYST family HAT inhibitor (MacPherson et al., 2020), compared to other MLL fusion-leukemia cells (Figure 6—figure supplement 1E), suggesting that HBO1-mediated protein acetylation may enhance the ELL-AF4 association. In summary, these results suggested that MLL-ELL transforms HPCs via interaction with AF4 family proteins, which is promoted by the HBO1 complex but hindered by other ELL-associated factors.

NUP98-HBO1 fusion transforms myeloid progenitors through interaction with MLL

NUP98-HBO1 fusion has been found in chronic myelomonocytic leukemia and was shown to induce clinically relevant leukemia in mice (Hayashi et al., 2019). NUP98-HBO1 transformed HPCs ex vivo, which was accompanied by high Hoxa9 expression (Figure 7A). Fusion of NUP98 and homeodomain proteins (e.g., NUP98-HOXA9) has been shown to induce leukemia in mouse models (Kroon et al., 2001). The homeodomain of HOXA9 possesses a sequence-specific DNA-binding ability. ChIP-qPCR analysis demonstrated that FLAG-tagged NUP98-HOXA9 (fNUP98-HOXA9) bound to the HOXA9-binding site within the HOXA7 locus, whereas fNUP98-HBO1 did not (Figure 7—figure supplement 1A), suggesting that HBO1 provides an alternative chromatin targeting function that differs from that of the HOXA9 homeodomain. Because the HBO1 complex binds MLL (Figure 7B), we hypothesized that the HBO1 portion might confer a targeting ability through association with MLL. To test this hypothesis, we generated an artificial NUP98 fusion construct in which MENIN is fused to NUP98 and examined its transforming properties. Indeed, NUP98-MENIN transformed HPCs (Figure 7C). Artificial NUP98 constructs fused with an HBO complex component (i.e., fNUP98-MEAF6 or -ING5) also transformed HPCs. IP-western blotting showed that these artificial NUP98 fusions were associated with MLL in the chromatin fraction, supporting the hypothesis that NUP98-HBO1 transforms HPCs via interaction with MLL (Figure 7D). The NUP98 portion did not co-precipitate MLL in this setting (Figure 7D, see fNUP98ΔFP), consistent with a previous report (Shima et al., 2017). However, it should be noted that the NUP98 portion was shown to localize in proximity with MLL (Xu et al., 2016) and promote the physical interaction of NUP98-HOXA9 with MLL (Shima et al., 2017). The sgRNA competition assay demonstrated fNUP98-HBO1-ICs depended on MLL for continuous proliferation (Figure 7E), similarly to fNUP98-HOXA9-ICs as reported previously (Shima et al., 2017; Xu et al., 2016). However, both fNUP98-HOXA9- and fNUP98-HBO1-ICs were mildly sensitive to MENIN-MLL interaction inhibitor (MI-2–2) (Shi et al., 2012), and were not rendered completely differentiated unlike MLL-AF10-ICs at the concentration of 10 μM (Figure 7F), suggesting that a MENIN-less MLL complex may sufficiently recruit NUP98-HBO1 to the target chromatin. Interestingly, E508Q amino acid substitution, which kills its HAT activity (Foy et al., 2008) but retains the binding capacities to MLL (Figure 7D), did not impair the transforming property of NUP98-HBO1 (Figure 7C). Moreover, WM1119 did not induce complete differentiation of NUP98-HBO1-ICs, as seen in MLL-AF10-ICs, at 10 μM (Figure 7—figure supplement 1B). These results indicate that the interaction with MLL, and not the intrinsic HAT activity, mediates NUP98-HBO1-mediated leukemic transformation.

Figure 7 with 1 supplement see all
Nucleoporin-98 (NUP98)-HBO1 fusion transforms myeloid progenitors through interaction with MLL.

(A) Structural requirement of NUP98-HBO1 fusion. Various NUP98 fusion constructs were examined for the transformation of myeloid progenitors (along with an HBO1 construct) as in Figure 1A. Relative Hoxa9 expression in first round colonies was analyzed. Asterisk indicates that the qPCR probe detected a human HOXA9 coding sequence included in the NUP98-HOXA9 construct in addition to endogenous murine Hoxa9. Zn: zinc finger; MYST: MYST HAT domain; HD: homeodomain; FG: phenylalanine-glycine repeat. (B) Association with MLL by NUP98-HBO1. The chromatin fraction of virus-packaging cells transiently expressing the indicated transgenes was subjected to immunoprecipitation (IP)-western blotting. Endogenous MLLC fragment was detected by a specific anti-MLL antibody. (C) Structural requirement of various NUP98 fusions for leukemic transformation. Indicated NUP98 fusion constructs were examined for the transformation of myeloid progenitors as in Figure 1A. ING: inhibitor of growth domain of ING4 and ING5; NuA4: histone acetyltransferase subunit NuA4; EPcA-I/II: enhancer of polycomb A domains I and II. (D) Association with MLL by various NUP98 fusions. IP-western blotting was performed as in (B). (E) Requirement of MLL for NUP98 fusion-immortalized cells. sgRNA competition assays for Kmt2a were performed on NUP98-HOXA9- and NUP98-HBO1-immortalized cells as in Figure 2F. (F) Effects of pharmacologic inhibition of MENIN-MLL interaction on NUP98 fusion-immortalized cells. NUP98-HBO1- and NUP98-HOXA9-immortalized cells were cultured in the presence of 10 μM of MI-2–2 MENIN-MLL interaction inhibitor, and their proliferation was monitored every 3 days. MLL-AF10-immortalized cells were also analyzed as a positive control. VC: vehicle control.

Association of MLL with the HBO1 complex is mainly mediated by PHF16 and ING4/5

To dissect the mechanism of association between MLL and the HBO1 complex, we performed IP-WB analyses of the chromatin fractions from HEK293T cells transiently expressing the MLL bait protein (fMLL 869–1124) in combination with each component of the HBO1 complex. Only PHF16 and ING5 were weakly detected in the co-precipitates of the MLL bait protein; however, a very strong association was observed when all of the HBO1 complex components were co-expressed (Figure 8A), suggesting that the association of MLL with the HBO1 complex is mediated via multiple contacts likely provided by PHF16 and ING5. IP-WB analysis wherein each component of the HBO1 complex was omitted indicated the PHF16 and ING5 are indeed the most influential components for the MLL-HBO1 complex association (Figure 8B). Notably, the omission of HBO1 reduced the association albeit to a much lesser extent, which can be rescued by adding back the C-terminal half of HBO1 that mediates interaction with PHF16 and ING5. The omission of MEAF6 also reduced the association substantially, indicating that the formation of the entire HBO1 complex maximizes the potential of the MLL-HBO1 complex association. Deletion of the ING domain from ING5 resulted in the loss of the MLL-HBO1 complex association, suggesting that the ING domain may provide a direct contact for MLL interaction. Point mutations of the aromatic cage of the PHD finger of ING5 (Y188A and W211A) that directly binds to H3K4me3 marks (Champagne et al., 2008) resulted in the loss of the MLL-HBO1 complex association, which could be rescued by ING4 (Figure 8C), indicating that ING4/5-mediated chromatin binding to H3K4me3 marks is required for efficient MLL-HBO1 complex association. These results suggest that ING4/5-mediated chromatin binding precedes the MLL-HBO1 complex association, and the MLL and HBO1 complexes stably associate with each other on the chromatin containing H3K4me3 marks to promote subsequent AEP/SL1-mediated transcriptional activation by MLL fusions (Figure 8D).

MLL-HBO1 complex association is mediated by multiple contacts and in a chromatin-bound context containing H3K4 methylation.

(A) Association of the MLL bait protein with the HBO1 complex. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293T cells transiently expressing FLAG-tagged MLL 869/1124 construct and Xpress-tagged HBO1 complex components was performed. (B) Influence of each HBO1 complex component for MLL-HBO1 complex association. (C) Domain mapping of ING5 responsible for MLL-HBO1 complex association. (D) Models of MLL-ELL- and nucleoporin-98 (NUP98)-HBO1-mediated gene activation.

Discussion

Most MLL fusion proteins constitutively recruit AEP by a variety of mechanisms to immortalize HPCs (Takahashi and Yokoyama, 2020). However, it was unclear how MLL-ELL exerts its oncogenic properties, as it was reported that an artificial MLL-EAF1 fusion immortalized HPCs (Luo et al., 2001). AF4 and EAF family proteins share structural similarities, both of which have the SDE motif and are shown to bind to SL1 (Figure 5A–COkuda et al., 2016). We previously demonstrated that the combination of SDE and NKW motifs is required for transcriptional activation (Okuda et al., 2015). Because EAF1 does not have an NKW motif, EAF1 is incompetent for transcriptional activation, and therefore MTMh-EAF1-C did not transform HPCs (Figure 5G). Instead, EAF1 homodimerizes through its EHD. The MTMT-EHD fusion (e.g., MTMTh-EAF1-N) transformed HPCs in a THD2-dependent manner, similar to MLL-AF6, which is known to transform HPCs via homodimerization (Liedtke et al., 2010). Thus, we concluded that MLL-EAF1 transforms HPCs via homodimerization. The S600A/K606T double mutation on ELL completely abrogated the interaction with EAF1, while retaining some binding capacity to AF4 and did not impair MLL-ELL-mediated transformation. Thus, we concluded that MLL-ELL transforms HPCs via association with AF4 family proteins, but not with EAF1 (Figure 8D).

ChIP-qPCR analysis indicated that ELL recruited AF4 and ENL to the target chromatin but was unable to further recruit SL1 and MED26 (Figure 5D), suggesting that the AEP complex must dissociate from ELL to function as a transcriptional activator (Figure 5E). Thus, MLL-ELL is a loading factor of the AEP complex, but not a nucleating factor (like MLL-ENL). In our previous study, we showed that MLL-AF10 is also an AEP loading factor that loads ENL onto chromatin (Okuda et al., 2017). It remains unclear how MLL-AF6, a dimer type of MLL fusion, affects the function of AEP and transforms HPCs. Nonetheless, ChIP-qPCR analysis showed that MLL-AF6 colocalized with AEP components at its target promoters (Yokoyama et al., 2010), suggesting that MLL-AF6 also loads AEP to the target chromatin via unknown mechanisms. All of the presumed AEP loading factor type MLL fusions (i.e., MLL-ELL, MLL-AF10, and MLL-AF6) showed susceptibility to Mllt1 knockdown, whereas the AEP nucleating factor type MLL fusions (e.g., MLL-ENL) were relatively resistant (Figure 5—figure supplement 1BOkuda et al., 2017; Yokoyama et al., 2010). Moreover, the AEP loading factor type MLL fusions exhibited relatively strong dependency on THD2, which mediates HBO1 complex recruitment (Figure 1). These results suggest that the HBO1 complex promotes loading of the AEP complex onto target chromatin (Figure 8D). Hence, the inhibitors that interfere with the MLL-HBO complex interaction may be highly effective for the AEP loading factor type MLL fusions.

Like MLL, NUP98 fuses with a variety of partners (Gough et al., 2011). NUP98 fusion partners can be roughly subdivided into three groups: the homeodomain-containing protein type (e.g., NUP98-HOXA9), the chromatin reader type (e.g., NUP98-HBO1, -LEDGF, -MLL, -NSD1, -PHF23, -BPTF, and -KDM5A), and the coiled-coil structure containing protein type. Among the chromatin reader type NUP98 fusions, MLL and LEDGF are components of the MLL complex (Yokoyama and Cleary, 2008), which targets previously transcribed CpG-rich promoters through association with unmethylated CpGs, di/tri-methylated histone H3 lysine 36 (H3K36me2/3) (Okuda et al., 2014), and di/tri-methylated histone H3 lysine 4 (H3K4me2/3) recognized by the PHD finger 3 of MLL (Milne et al., 2010; Wang et al., 2010). NUP98-NSD1 was shown to activate HOX-A genes through the association with methylated H3K36 marks (Wang et al., 2007), and NUP98-PHF23, -BPTF, and-KDM5A were through association with H3K4me2/3 marks (Zhang et al., 2020). Thus, the chromatin reader type NUP98 fusions confer a targeting ability to the chromatin of the HOX-A loci by interacting with specific chromatin modifications or chromatin-binding proteins. A subset of NUP98 fusions including NUP98-HBO1 and -LEDGF appear to target through interaction with MLL at least in part. This was supported by the immortalization of HPCs by the artificial fusion of NUP98 and MENIN (Figure 7C). It has been reported that a portion of MLL target genes is regulated in a MENIN-independent manner (Artinger et al., 2013) and an MLL mutant protein lacking the MENIN-binding motif but retaining the CXXC domain and the PHD finger 3 localized at the HOXA9 locus presumably through the association with unmethylated CpGs and H3K4me2/3 (Milne et al., 2010; Wang et al., 2010), suggesting that MLL can bind to some of its target chromatin in a MENIN-independent manner. The NUP98-MLL fusion protein expressed in leukemia patients does not retain the structures responsible for interaction with MENIN and LEDGF (Kaltenbach et al., 2010), suggesting that the MLL portion in NUP98-MLL confers its targeting ability in a MENIN-independent manner. The proliferation of NUP98-HBO1-ICs was dependent on MLL (Figure 7E) and was slowed but not entirely blocked by the MENIN-MLL interaction inhibitor (Figure 7F). These results indicate that a MENIN-less MLL complex may sufficiently recruit NUP98-HBO1 possibly through the association with H3K4me3 marks via the PHD finger 3 of MLL. The presence of H3K4me3 marks at the target promoter is also a prerequisite for the HBO1 complex-MLL association on chromatin as they are recognized by the PHD finger of ING4/5 subunits (Figure 3F and Figure 8CChampagne et al., 2008). Taken together, these results suggest that a subset of NUP98 fusions target the HOX-A loci via association with MLL and H3K4me3 marks and activate transcription by NUP98-mediated functions (Figure 8D).

This study demonstrated that various MLL fusions and NUP98 fusions transform HPCs via HBO1-MLL interaction (Figure 8D). Hence, we propose that the binding modules in this interaction can be a good molecular target for drug development. HBO1-MLL interaction inhibitors would complement the therapeutic effects of the emerging MENIN-MLL interaction inhibitors (Klossowski et al., 2019; Krivtsov et al., 2019) to treat malignant leukemia with subsets of MLL and NUP98 fusions.

Materials and methods

Materials availability

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Materials generated in this study will be provided upon request.

Experimental model and subject details

Vector constructs

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For protein expression vectors, cDNAs obtained from Kazusa Genome Technologies Inc (Nagase et al., 2008) were modified by PCR-mediated mutagenesis and cloned into the pMSCV vector (for virus production) or pCMV5 vector (for transient expression) by restriction enzyme digestion and DNA ligation. The MSCV-neo MLL-ENL and MLL-AF10 vectors have been previously described (Okuda et al., 2017). sgRNA expression vectors were constructed using the pLKO5.sgRNA.EFS.GFP vector (Heckl et al., 2014). shRNA expression vectors were constructed using a pLKO.1 vector or were purchased from Dharmacon. The target sequences of sgRNA are listed in Supplementary file 1a and b.

Cell lines

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HEK293T cells were a gift from Michael Cleary and were authenticated by the JCRB Cell Bank in 2019. HEK293TN cells were purchased from System Biosciences. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (PS). The Platinum-E (PLAT-E) ecotropic virus-packaging cell line—a gift from Toshio Kitamura (Morita et al., 2000)—was cultured in DMEM supplemented with 10% FBS, puromycin, blasticidin, and PS. The human leukemia cell lines HB1119 and REH, gifts from Michael Cleary (Tkachuk et al., 1992), were cultured in RPMI 1640 medium supplemented with 10% FBS and PS. HEK293T–LUC cells were generated by transduction of the lentivirus carrying pLKO1-puro-FR-LUC, as described previously (Okuda et al., 2015). Murine myeloid progenitors immortalized by various transgenes were cultured in RPMI 1640 medium supplemented with 10% FBS, and PS containing murine stem cell factors (mSCFs), murine interleukin-3 (mIL-3), and murine granulocyte-macrophage colony-stimulating factor (mGM-CSF; 1 ng/mL of each). Cells were cultured in the incubator at 37°C and 5% CO2 and routinely tested for mycoplasma using the MycoAlert Mycoplasma detection kit (Lonza).

Western blotting

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Western blotting was performed as previously described (Yokoyama et al., 2002). Briefly, proteins were separated electrophoretically in an acrylamide gel and were transblotted onto nitrocellulose sheets using a mini transblot cell (Bio-Rad). The nitrocellulose sheets were blocked with 5% skimmed milk in T-PBS (phosphate-buffered saline containing 0.1% Tween 20) for 1 hr, rinsed twice with T-PBS, and incubated with primary antibodies suspended in 5% skim milk in T-PBS overnight. The blots were then washed twice with T-PBS and incubated with peroxidase-conjugated secondary antibodies for 2 hr. Chemiluminescence was performed using the ECL chemiluminescence reagent (GE Healthcare). The antibodies used in this study are listed in Key resources table.

Virus production

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Ecotropic retrovirus was produced using PLAT-E packaging cells (Morita et al., 2000). Lentiviruses were produced in HEK293TN cells using the pMDLg/pRRE, pRSV-rev, and pMD2.G vectors, all of which were gifts from Didier Trono (Dull et al., 1998). The virus-containing medium was harvested 24–48 hr following transfection and used for viral transduction.

Myeloid progenitor transformation assay

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The myeloid progenitor transformation assay was carried out as previously described (Lavau et al., 1997; Okuda and Yokoyama, 2017b). Bone marrow cells were harvested from the femurs and tibiae of 5-week-old female C57BL/6J mice. c-Kit-positive cells were enriched using magnetic beads conjugated with an anti-c-Kit antibody (Miltenyi Biotec), transduced with a recombinant retrovirus by spinoculation, and then plated (4 × 104 cells/sample) in a methylcellulose medium (Iscove’s modified Dulbecco’s medium, 20% FBS, 1.6% methylcellulose, and 100 µM β-mercaptoethanol) containing mSCF, mIL-3, and mGM-CSF (10 ng/mL each). During the first culture passage, G418 (1 mg/mL) or puromycin (1 µg/mL) was added to the culture medium to select the transduced cells. Hoxa9 expression was quantified by qRT-PCR after the first passage. Cells were then re-plated once every 5 days with fresh medium. Colony-forming units were quantified per 104 plated cells at each passage.

In vivo leukemogenesis assay

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In vivo leukemogenesis assay was carried out as previously described (Lavau et al., 1997; Okuda and Yokoyama, 2017a). Cells positive for c-Kit (2 × 105), prepared from mouse femurs and tibiae, were transduced with retrovirus by spinoculation and intravenously transplanted into sublethally irradiated (5–6 Gy) 8-week-old female C57BL/6JJcl (C57BL/6J) mice. Moribund mice were euthanized, and the cells isolated from their bone marrow were cultured in methylcellulose medium used for myeloid progenitor transformation assays for more than three passages to remove untransformed cells and then subjected to secondary transplantation. For secondary leukemia, leukemia cells (2 × 105) cultured ex vivo were transplanted in the same manner as the primary transplantation. This protocol was approved by the National Cancer Center Institutional Animal Care and Use Committee.

qRT-PCR

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Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse-transcribed using the Superscript III First Strand cDNA Synthesis System (Thermo Fisher Scientific) with oligo (dT) primers. Gene expression was analyzed by qPCR using TaqMan probes (Thermo Fisher Scientific). Relative expression levels were normalized to those of GAPDH/Gapdh and determined using a standard curve and the relative quantification method, according to the manufacturer’s instructions (Thermo Fisher Scientific). Commercially available PCR probe sets used in this study are listed in Key resources table.

Fractionation-assisted chromatin immunoprecipitation

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Chromatin fractions from HEK293T and HB1119 cells were prepared using the fanChIP method, as described previously (Miyamoto and Yokoyama, 2021; Okuda et al., 2014). Cells were suspended in CSK buffer (100 mM NaCl, 10 mM PIPES [pH 6.8], 3 mM MgCl2, 1 mM EGTA, 0.3 M sucrose, 0.5% Triton X-100, 5 mM sodium butyrate, 0.5 mM DTT, and protease inhibitor cocktail) and centrifuged (400× g for 5 min, at 4°C) to remove the soluble fraction. The pellet was resuspended in MNase buffer (50 mM Tris-HCl [pH 7.5], 4 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 5 mM sodium butyrate, 0.5 mM DTT, and protease inhibitor cocktail) and treated with MNase at 37°C for 3–6 min to obtain oligonucleosomes. MNase reaction was then stopped by adding EDTA (pH 8.0) to a final concentration of 20 mM. An equal amount of lysis buffer (250 mM NaCl, 20 mM sodium phosphate [pH 7.0], 30 mM sodium pyrophosphate, 5 mM EDTA, 10 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM DTT, and EDTA-free protease inhibitor cocktail) was added to increase solubility. The chromatin fraction was cleared by centrifugation (15,000 rpm for 5 min, 4°C) and subjected to immunoprecipitation with specific antibodies and Protein-G magnetic microbeads (Invitrogen) or with anti-FLAG M2 antibody-conjugated beads (Key resource table). Immunoprecipitates were then washed five times with washing buffer (1:1 mixture of lysis buffer and MNase buffer with 20 mM EDTA) and eluted in elution buffer (1% SDS and 50 mM NaHCO3). The eluted material was analyzed by multiple methods including western blotting, qPCR, and deep sequencing. Optionally, the immunoprecipitates were washed twice with washing buffer, twice with MNase buffer, and then treated with DNase I (Qiagen) for 15 min at room temperature. The immunoprecipitates were further washed four times with washing buffer and eluted using elution buffer. The eluted materials were subjected to western blotting and SYBR green staining.

ChIP-qPCR and ChIP-seq

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The eluted material obtained by fanChIP was extracted using phenol/chloroform/isoamyl alcohol. DNA was precipitated with glycogen, dissolved in TE buffer, and analyzed by qPCR (ChIP-qPCR) or deep sequencing (ChIP-seq). The qPCR probe/primer sequences are listed in Supplementary file 1c. For deep sequencing, DNA was further fragmented (~150 bp) using the Covaris M220 DNA shearing system (M and M Instruments Inc). Deep sequencing was then performed using the TruSeq ChIP Sample Prep Kit (Illumina) and HiSeq2500 (Illumina) at the core facility of Hiroshima University. Data were visualized using Integrative Genome Viewer (The Broad Institute). Raw reads in FASTQ format were trimmed using Cutadapt and aligned to the reference genome hg19 with BWA (Li and Durbin, 2009; Martin, 2011). Accession numbers and sample IDs are listed in Supplementary file 1d.

Liquid chromatography-tandem mass spectrometry analysis

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Proteins were digested with trypsin and tandem mass spectrometry was performed using an LTQ Orbitrap ELITE ETD mass spectrometer (Thermo Fisher Scientific), as described previously (Okuda et al., 2017).

sgRNA competition assay

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Cas9 was introduced via lentiviral transduction using the pKLV2-EF1a-Cas9Bsd-W vector (Tzelepis et al., 2016). Cas9-expressing stable lines were established with blasticidin (10–30 µg/mL) selection. The sequences of sgRNAs are listed in Supplementary file 1a and b. The targeting sgRNA was co-expressed with GFP via lentiviral transduction using pLKO5.sgRNA.EFS.GFP vector (Heckl et al., 2014). Percentages of GFP-positive cells were initially determined by FACS analysis at 2 days after sgRNA transduction and then measured every 4 days.

Accession numbers

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Deep sequencing data used in this study have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under the accession numbers listed in Supplementary file 1d (ChIP-seq, CIRA-seq, and RNA-seq). The mass spectrometry data were deposited to Japan ProteOme STandard Repository (jPOSTrepo) under the accession number JPST001262 (PXID:PXD027400).

Statistics

Statistical analysis was performed using GraphPad Prism7 software. Data are presented as the mean with standard deviation. Multiple comparisons were performed by two-way ANOVA. The statistical tests were two-sided. Mice transplantation experiments were analyzed by the log-rank test and Bonferroni correction was applied for multiple comparisons. p-values < 0.05 were considered statistically significant. n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Study approval

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All animal experimental protocols were approved by the National Cancer Center (Tokyo, Japan) Institutional Animal Care and Use Committee (T17501).

Appendix 1

Appendix 1—key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
OtherMNaseSigma-AldrichCat#:N3755-200U
OtherProtein-G Magnetic beadsThermo Fisher ScientificCat#:1004D
OtherFLAG M2 antibody-conjugated beadsSigma-AldrichCat#:M8823
RRID:AB_2637089
Otherc-Kit magnetic beadsMiltenyi BiotecCat#:130-091-224
RRID:AB_2753213
OtherMI-2–2CalbiochemCat#:444825
OtherWM1119EnamineCat#:EN300-1719156
AntibodyMLL(N)Cell Signaling TechnologyCat#:14689
RRID:AB_2688009
WB(1000:1)
ChIP(1 μL/400 μL)
AntibodyMLL(N)Generated in-house Yokoyama et al., 2002rpN1ChIP(1 μg/400 μL)
AntibodyMOZActive motifCat#:39868ChIP(1 μg/400 μL)
AntibodyAF4Santa Cruz BiotechnologyCat#:sc-49350
RRID:AB_2226113
ChIP(1 μg/400 μL)
AntibodyTAF1CBethyl LaboratoriesCat#:A303-698A
RRID:AB_11203194
ChIP(1 μg/400 μL)
AntibodyAF17Bethyl LaboratoriesCat#:A302-198A
RRID:AB_1659777
ChIP(1 μg/400 μL)
AntibodyDOT1LBethyl LaboratoriesCat#:A300-953A
RRID:AB_805775
ChIP(1 μg/400 μL)
AntibodyCyclinT1Santa Cruz BiotechnologyCat#:sc-8127
RRID:AB_2073892
ChIP(1 μg/400 μL)
AntibodyENLCell Signaling TechnologyCat#:14893SWB(1000:1)
ChIP(5 μL/400 μL)
AntibodyHistone H3K4me3Active motifCat#:39159
RRID:AB_2615077
ChIP(1 μg/400 μL)
AntibodyHistone H3K18acAbcamCat#:ab1191
RRID:AB_298692
ChIP(1 μg/400 μL)
AntibodyRNAP2 non-PAbcam8WG16/Cat#:ab817
RRID:AB_306327
ChIP(1 μg/400 μL)
AntibodyRNAP2 Ser5-PMilliporeCat#:05–623
AB_309852
ChIP(1 μg/400 μL)
AntibodyFLAGSigma-AldrichCat#:F3165
RRID:AB_259529
ChIP(1 μg/400 μL)
AntibodyFLAGSigma-AldrichCat#:F7425
RRID:AB_439687
WB(1000:1)
AntibodyHARoche3F10/Cat#:11867423001
RRID:AB_390918
WB(1000:1)
ChIP(0.2 μg/400 μL)
AntibodyXpressSanta Cruz BiotechnologyCat#:sc-7270
RRID:AB_675763
WB(1000:1)
ChIP(1 μg/400 μL)
AntibodyELLCell Signaling TechnologyCat#:14468S
RRID:AB_2798489
WB(1000:1)
AntibodyHBO1AbcamCat#:70183
RRID:AB_1269226
WB(1000:1)
ChIP(1 μg/400 μL)
AntibodyPHF16AbcamCat#:129495
RRID:AB_11157084
WB(1000:1)
ChIP(1 μg/400 μL)
AntibodyMEAF6STJCat#:116836WB(1000:1)
ChIP(1 μg/400 μL)
AntibodyING4AbcamCat#:108621
RRID:AB_10860023
WB(1000:1)
ChIP(1 μg/400 μL)
AntibodyMLL(C)Cell Signaling TechnologyCat#:14197S
RRID:AB_2688010
WB(1000:1)
AntibodyAF4Bethyl LaboratoriesCat#:A302-344A
RRID:AB_1850255
WB(1000:1)
AntibodyAF5Q31Bethyl LaboratoriesCat#:A302-538A
RRID:AB_1998985
WB(1000:1)
AntibodyP53Santa Cruz BiotechnologyCat#:sc-126
RRID:AB_628082
WB(1000:1)
Sequence-based reagentGAPDHThermo Fisher ScientificHs02786624_g1
Sequence-based reagentHOXA9Thermo Fisher ScientificHs00365956_m1
Sequence-based reagentMYCThermo Fisher ScientificHs01570247_m1
Sequence-based reagentRPL13AThermo Fisher ScientificHs04194366_g1
Sequence-based reagentCDKN2CThermo Fisher ScientificHs00176227_m1
Sequence-based reagentGapdhThermo Fisher ScientificMm99999915_g1
Sequence-based reagentHoxa9Thermo Fisher ScientificMm00439364_m1
Sequence-based reagentMllt1Thermo Fisher ScientificMm00452080_m1
Sequence-based reagentsgRNAsThis studySee Supplementary file 1
Sequence-based reagentCustom made qPCR primersThermo Fisher ScientificSee Supplementary file 1
Recombinant DNA reagentpMSCV-neoClontechCat#:634401
Recombinant DNA reagentpLKO5.EFS.GFPAddgene (gift from Benjamin Ebert) Heckl et al., 2014Addgene #57822
RRID:Addgene_57822
Recombinant DNA reagentpKLV2-Cas9.bsdAddgene (gift from Kosuke Yusa) Tzelepis et al., 2016Addgene #68343
RRID:Addgene_68343
Recombinant DNA reagentpMDLg/pRREAddgene (gift from Didier Trono) Dull et al., 1998Addgene #12251
RRID:Addgene_12251
Recombinant DNA reagentpRSV-revAddgene (gift from Didier Trono) Dull et al., 1998Addgene #12253
RRID:Addgene_12253
Recombinant DNA reagentpMD2.GAddgene (gift from Didier Trono) Dull et al., 1998Addgene #12259
RRID:Addgene_12259
Recombinant DNA reagentpLKO.1-puroAddgene (gift from Bob Weinberg) Stewart et al., 2003Addgene #8453
RRID:Addgene_84530
Recombinant DNA reagentpLKO.1-sh-Mllt1-puroGE HealthcareTRCN0000084405
Software, algorithmGraphPad Prism7GraphPad Software IncRRID:SCR_002798
Software, algorithmIntegrative Genomics ViewerThorvaldsdóttir et al., 2013RRID:SCR_011793
Software, algorithmCutadaptMartin, 2011RRID:SCR_011841
Software, algorithmBWALi and Durbin, 2009RRID:SCR_010910
Cell line (Homo sapiens)Human: HEK293TGift from Michael ClearyAuthenticated by JCRB Cell Bank in 2019
Cell line (Homo sapiens)Human: HB1119Gift from Michael Cleary Tkachuk et al., 1992The cell line was verified by the expression of MLL-ENL
Cell line (Homo sapiens)Human: REHGift from Michael Cleary Yokoyama et al., 2004RRID:CVCL_1650Authenticated by JCRB Cell Bank in 2021
Cell line (Homo sapiens)Human: PLAT-EGift from Toshio Kitamura Morita et al., 2000RRID:CVCL_B488
Cell line (Homo sapiens)Human: HEK293TNSystem BioscienceCat#:LV900A-1
RRID:CVCL_UL49
Cell line (Homo sapiens)Human: HEK293T dMLLc3Generated in-house Miyamoto et al., 2020
Cell line (Homo sapiens)Human: HEK293T dMLLc7Generated in-house Miyamoto et al., 2020
Cell line (Homo sapiens)Human: HEK293TLGenerated in-house Okuda et al., 2015
Strain, strain background (Mus musculus)Mouse: C57BL/6JCLEA Japan

Data availability

ChIP-seq data, CIRA-seq data, and RNA-seq data have been deposited at the DDBJ (DNA Data Bank of Japan) Sequence Read Archive under the accession numbers (DRA010818, DRA004871, DRA004872, DRA010819, DRA008732, DRA008734, and DRA004874) and sample IDs listed in Supplementary file 1.The mass spectrometry data were deposited to Japan ProteOme STandard Repository (jPOSTrepo) under the accession number JPST001262 (PXID:PXD027400).

The following data sets were generated
    1. HIROSHIMA
    (2020) DDBJ
    ID DRA010818. Genomic localization of various factors/modifications in HB1119 cells.
    1. HIROSHIMA
    (2020) DDBJ
    ID DRA010819. Genomic localization of various FLAG-tagged proteins in HEK293T cells.
    1. HIROSHIMA
    (2021) DDBJ
    ID DRA012472. Genomic localization of various proteins in HB1119 cells.
    1. HIROSHIMA
    (2021) DDBJ
    ID DRA012473. Genomic localization of various proteins in HEK293T cells.
The following previously published data sets were used
    1. HIROSHIMA
    (2016) DDBJ
    ID DRA004871. Genomic localization of various factors/modificaions in HB1119 cells.
    1. HIROSHIMA
    (2016) DDBJ
    ID DRA004872. Genomic localization of various factors/modificaions in 293T cells.
    1. HIROSHIMA
    (2019) DDBJ
    ID DRA004874. Expression profiles of HB1119 and 293T cell lines.
    1. HIROSHIMA
    (2019) DDBJ
    ID DRA008732. ChIP-seq analysis of HEK293T cell lines.
    1. HIROSHIMA
    (2019) DDBJ
    ID DRA008734. CIRA-seq analysis of the HEK293T cell line.

References

Decision letter

  1. Xiaobing Shi
    Reviewing Editor; Van Andel Institute, United States
  2. Kevin Struhl
    Senior Editor; Harvard Medical School, United States
  3. Xiaobing Shi
    Reviewer; Van Andel Institute, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This is a quite comprehensive study demonstrating that various MLL fusions transform hematopoietic progenitors via HBO1-MLL interaction. The additional data provided in the revised manuscript have addressed most prior questions. This study adds mechanistic depth into the important recent discovery of HBO1 functions in MLL-fusion leukemias and provides potentially new therapeutic approach.

Decision letter after peer review:

Thank you for submitting your article "HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Xiaobing Shi as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The manuscript by Takahashi et al., describes the interaction between MLL fusion proteins with HBO1 and its role in leukemogenesis. Myeloid progenitor transformation assays using various MLL fusion proteins reveal that MLL fusion proteins requires the TRX2 domain of MLL for effective leukemic transformation. IP-MS identifies HBO1 as a bona fide binding partner of the MLL TRX2 domain. ChIP-seq experiments in various cancer cell lines indicate overlap between MLL and some of the HBO complex subunits, however this does not establish recruitment at these sites. ChIP-qPCR at a few MLL-fusion target genes with MLL depletion supports the recruitment hypothesis somewhat although with only mixed and modest effect sizes. At this point the paper shifts to a seemingly distinct line of inquiry, which is not closely related to the HBO1-TRX2 story to the first three figures. The new direction examines the ELL fusion partner in some detail using similar fusion protein chimeras, but a portion of Figure 4, is largely confirmatory of previously established findings about the critical regions of ELL for transformation and its AF4/EAF1 partners, adding only that portions of the MLL fusion protein are dispensable, provided that they are replaced with the PWWP of LEDGF. Further experiments define potentially two distinct complexes that have already been characterized being recruited by ELL, although there is overlap here again with their previous studies, and the results are a little hard to interpret. The last figure in the paper tackles the seemingly unrelated Nup98-HBO1 fusion, a rare patient mutation-they demonstrate a requirement for MLL for viability of hematopoietic progenitors transformed by this fusion, connecting back to the TRX2 interaction, and show that Menin inhibitors slow growth.

Overall, this is a quite comprehensive study demonstrating that various MLL fusions transform hematopoietic progenitors via HBO1-MLL interaction. The quality of the science fairly high (with a few noted exceptions that can be addressed), but the lack of clarity in presentation actively obscures really nice and seemingly important elements of this work. It also stops short of really demonstrating that the MLL-TRX2 motif recruits HBO1 in some direct way, which would be of high impact if it could be more clearly molecularly defined.

1. The most significant impact of this manuscript is the identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex, which adds mechanistic depth to the important recent discovery that the MLL-fusion leukemias rely on HBO1 function (MacPherson et al., Nature 2020). However, it is not clear whether this interaction is direct, or bridged by some, as yet unidentified factor including DNA. It is also important to define the point of contact on the HBO1 side (even which subunit).

2. The current evidence does not conclusively demonstrate that The MLL-HBO1 interaction is the mechanism of HBO1 recruitment. It would be helpful to do a ChIP experiment in the MLL null background rescuing with +/- TRX2 to demonstrate that this "domain" is responsible for the recruitment-- even more ideal, but perhaps beyond the scope would be a point-mutant that disrupts direct contact to the HBO1 complex.

3. A large number of ChIP-seq and RNA-seq are performed. However, it is not clear how many repeats were performed. This need to be clearly stated, and biological repeats are needed for at least some of the essential ones, such as MLL1, HBO1 and H3K14ac, the mark deposited by HBO1. It is also important to describe clearly the source and specificity of the antibodies used for ChIP.

4. It seems that characterization of the AF4 and EAF1 associated complexes (in Figures 4-6) is unrelated to the main topic of this paper. This part is essentially a reinterpretation of prior results from Okuda, JCI 2017. This portion of the prior paper should be corrected there.

https://doi.org/10.7554/eLife.65872.sa1

Author response

Essential revisons:

1. The most significant impact of this manuscript is the identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex, which adds mechanistic depth to the important recent discovery that the MLL-fusion leukemias rely on HBO1 function (MacPherson et al., Nature 2020). However, it is not clear whether this interaction is direct, or bridged by some, as yet unidentified factor including DNA. It is also important to define the point of contact on the HBO1 side (even which subunit).

To determine the major points of contact, we performed an immunoprecipitation followed by western blotting (IP-WB) analysis of the chromatin fractions from HEK293T cells transiently expressing various components of the HBO1 complex and the MLL bait protein (Figure 8). When each HBO1 complex component was co-expressed with the MLL bait protein, PHF16 and ING5 were weakly detected in the coprecipitates of the MLL bait protein, whereas HBO1 and MEAF6 were not (Figure 8A), suggesting that PHF16 and ING5 provide major points of contact for MLL. When all of the HBO complex components were co-expressed with the MLL bait protein, a very strong association was observed (Figure 8A, see fMLL 869/1124 + HBO1 complex), suggesting that MLL associates with the HBO1 complex through multiple contacts. Next, we omitted one component each from the HBO1 complex and tested which component provides the most significant contact for MLL. Omission of PHF16 or ING5 resulted in a drastic decrease of MLL-HBO1 complex association (Figure 8B), supporting the notion that PHF16 and ING5 provide major points of contact. Indeed, NUP98-ING5 fusion co-IPed MLL more efficiently compared to NUP98-HBO1 and NUP98-MEAF6 (Figure 7D). To further narrow down the structures responsible for MLL-HBO complex interaction, we generated a series of ING5 mutants and tested their association with MLL. Deleting the ING domain from ING5 resulted in the loss of MLL interaction (Figure 8C). In addition, mutations in the aromatic cage of the PHD finger of ING5, which is critical for methylation-specific association with Histone H3K4, also resulted in the loss of MLL interaction. This suggests that ING5 needs to be bound to methylated histone H3K4 and possibly mediates direct interaction with MLL via its ING domain. These results indicate that MLL-HBO1 complex interaction is mediated by multiple points of contact mainly mediated by PHF16 and ING5 in the chromatin-bound form. These new observations/notions have been added to the revised manuscript. Overall, our results indicated that MLL-HBO1 complex formation is highly context-dependent, which preferentially occurs on chromatin as a fully assembled HBO1 complex. As DNAseI treatment did not hinder the interaction, we believe that DNA is not required for MLL-HBO1 interaction (Figure 2—figure supplement 2B).

2. The current evidence does not conclusively demonstrate that The MLL-HBO1 interaction is the mechanism of HBO1 recruitment. It would be helpful to do a ChIP experiment in the MLL null background rescuing with +/- TRX2 to demonstrate that this "domain" is responsible for the recruitment-- even more ideal, but perhaps beyond the scope would be a point-mutant that disrupts direct contact to the HBO1 complex.

We have provided ChIP-qPCR analysis data of HEK293T cells lacking MLL (Figure 3F). We performed ChIP for HBO1 using 3 biological replicates and observed a consistent decrease in the HBO1 ChIP signals at the MYC promoter, supporting the idea that MLL-HBO1 interaction is the mechanism of HBO1 recruitment, at least partially, at the MYC promoter. However, it is also true that HBO1 is recruited to other non-MYC promoters such as RPL13A in the absence of MLL, indicating that HBO1 is recruited to the promoter in non-MLL-mediated manners as well. H3K4me3 marks were substantially decreased at the MYC promoter but not at the RPL13A promoter in MLL-deficient cells. This suggested that MLL-mediated H3K4 methylation is required for ING4/5-mediated HBO1 complex recruitment. As advised by the reviewer, we examined the ability of MLL mutants, with or without THD2 (Formally termed as TRX2 domain), to recruit HBO1 in MLL null background. We did not observe any substantial increase of HBO1 recruitment by the THD2-containing MLL mutant (Figure 3—figure supplement 1C), indicating that THD2 is not sufficient for HBO1 recruitment but requires the pre-existing H3K4me3 marks. We think that the MLL-HBO1 interaction enhances the recruitment of HBO1 to certain promoters such as MYC, while the HBO1 can be recruited to promoters in other ways. We speculate that gene promoters which depend on the AEP/SL1-mediated transcriptional pathway are heavily dependent on the MLL-HBO1 complex. In accord, the MYC promoter shows a relatively robust decrease of the ChIP signals of AEP/SL1 (i.e., ENL, CCNT1, CDK9 and TAF1C) in MLL knockout cells (Figure 3 and Figure 3—figure supplement1B). In the revised manuscript, we have added these new findings and modified the text to avoid giving the misleading impression that MLL is the sole element whose presence is sufficient for the HBO1 complex recruitment.

3. A large number of ChIP-seq and RNA-seq are performed. However, it is not clear how many repeats were performed. This need to be clearly stated, and biological repeats are needed for at least some of the essential ones, such as MLL1, HBO1 and H3K14ac, the mark deposited by HBO1. It is also important to describe clearly the source and specificity of the antibodies used for ChIP.

We have performed ChIP-seq analyses of MLL (MLL1) and HBO1complex in duplicates and deposited in the public archive. ChIP-seq data of H3K14ac was not presented in the initial manuscript. The HBO1/BRPF2 complex was shown to be responsible for the global acetylation of H3K14ac in the hematopoietic cells (1). However, it is considered that the preferred histone modifications provided by the HBO1/PHF16 complex are histone H4K5/8/12 acetylations rather than H3K14/23 acetylations (2). We apologize for indicating that the HBO1/PHF16 complex provides H3K14ac marks in the cartoon in the previous manuscript, which was incorrect. Thus, PHF16-, MEAF6-, and ING4-ChIP analyses were performed to more clearly demonstrate the specific localization of the HBO1 complex and the results have been presented. Details of the antibodies used for ChIP experiments are described in the revised manuscript in Key resource table.

4. It seems that characterization of the AF4 and EAF1 associated complexes (in Figures 4-6) is unrelated to the main topic of this paper. This part is essentially a reinterpretation of prior results from Okuda, JCI 2017. This portion of the prior paper should be corrected there.

The main focus of this study was understanding the role of the HBO1 complex in MLL-mediated transactivation pathways. The structure/function analysis of MLL fusion proteins demonstrated that MLL-ELL is highly dependent on the HBO1-mediated function in leukemic transformation. Hence, it was important to clarify the mechanism of gene activation by MLL-ELL in this paper to understand why HBO1 association is critically required for MLL-ELL-mediated transformation. Because MLL-ELL associates with AEP similarly to other major MLL fusions such as MLL-AF4 and MLL-ENL, it was speculated that MLL-ELL also activates its target genes via AEP. However, ELL associates with EAF family proteins and MLL-EAF also has transforming ability (3), which contradicts this idea. Thus, EAF1-mediated function could be more important for MLL-ELL-mediated transformation. To dissect the molecular mechanism of transformation, we generated a point mutant that selectively impaired ELL-EAF interaction and demonstrated that EAF1-association is dispensable for MLL-ELL-mediated transformation (Figure 6), thereby indicating that MLL-ELL transforms via AEP-mediated functions, which demands HBO1-mediated functions. We also showed that the presence of THD2 enhances ELL-AEP association to further suggest that one of the roles for the HBO1 complex is to enhance the association of ELL with AEP. These findings are not reinterpretations of our prior results and are relevant to the main topic of this paper, and therefore we have retained them in the revised manuscript.

https://doi.org/10.7554/eLife.65872.sa2

Article and author information

Author details

  1. Satoshi Takahashi

    1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    2. Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan
    Contribution
    Conceptualization, Resources, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Akinori Kanai

    Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
    Contribution
    Resources, Data curation, Software, Formal analysis, Visualization, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1555-6768
  3. Hiroshi Okuda

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Resources, Formal analysis, Validation
    Competing interests
    No competing interests declared
  4. Ryo Miyamoto

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Yosuke Komata

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  6. Takeshi Kawamura

    Isotope Science Center, The University of Tokyo, Tokyo, Japan
    Contribution
    Data curation, Formal analysis, Visualization, Methodology
    Competing interests
    No competing interests declared
  7. Hirotaka Matsui

    Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
    Contribution
    Resources, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  8. Toshiya Inaba

    Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  9. Akifumi Takaori-Kondo

    Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7678-4284
  10. Akihiko Yokoyama

    1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    2. Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan
    3. Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    ayokoyam@ncc-tmc.jp
    Competing interests
    A.Y. received a research grant from Dainippon Sumitomo Pharma Co. Ltd.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5639-8068

Funding

Japan Society for the Promotion of Science (16H05337)

  • Akihiko Yokoyama

Japan Society for the Promotion of Science (19H03694)

  • Akihiko Yokoyama

Yamagata Health Support Association

  • Akihiko Yokoyama

the City of Tsuruoka

  • Akihiko Yokoyama

Leukemia Research Foundation

  • Akihiko Yokoyama

Dainippon Sumitomo Pharma Co., Ltd.

  • Akihiko Yokoyama

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Ikuko Yokoyama, Hagumu Sato, Kanae Ito, Makiko Okuda, Yuzo Sato, Megumi Nakamura, Etsuko Kanai, Aya Nakayama, and Ayako Yokoyama for technical assistance. We also thank the Shonai Regional Industry Promotion Center members for their administrative support. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants (16H05337, 19H03694 to AY). This work was also supported in part by research funds from the Yamagata prefectural government, the City of Tsuruoka, Dainippon Sumitomo Pharma Co. Ltd., and the Friends of Leukemia Research Fund.

Ethics

Animal experimentation: All animal experimental protocols were approved by the National Cancer Center (Tokyo Japan) Institutional Animal Care and Use Committee.

Senior Editor

  1. Kevin Struhl, Harvard Medical School, United States

Reviewing Editor

  1. Xiaobing Shi, Van Andel Institute, United States

Reviewer

  1. Xiaobing Shi, Van Andel Institute, United States

Publication history

  1. Received: December 17, 2020
  2. Preprint posted: January 8, 2021 (view preprint)
  3. Accepted: August 12, 2021
  4. Version of Record published: August 25, 2021 (version 1)

Copyright

© 2021, Takahashi et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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