MLL associates with a cohort of proteins shared with the yeast and human SET1 HMT complexes.
MLL and associated factors were biochemically purified by using conventional and affinity chromatography. Nuclear extract prepared from the K562 erythroleukemia cell line was subjected to Q-Sepharose (anion exchange) and heparin Sepharose chromatography and then was immunoabsorbed by using a monoclonal antibody specific for the p300 subunit of MLL (MLLN
) (Fig. ). Denaturing gel electrophoretic analysis of the MLLN
immunopurification product revealed the presence of eight polypeptides that were not present in control purifications with an unrelated monoclonal antibody (anti-SUV39H1) (Fig. ). A similar polypeptide profile was observed when the purification was performed with a monoclonal antibody specific for the p180 MLLC
subunit (Fig. ). The eight polypeptides were identified by mass spectrometry to be the processed portions of MLL (MLLN
) and six previously known but mostly uncharacterized proteins, including HCF-2, ASH2L1 and -2 (human homologs of Drosophila
), menin (gene product of MEN1
), RBBP5 (also called RBQ-3) (49
), and WDR5 (also called BIG-3) (20
FIG. 1. Purification of MLL and identification of associated proteins. (A) The scheme for purification of MLL complex indicates the chromatography and elution conditions employed. ppt, precipitate. (B) Purification of MLL complex. The protein eluted at 500 mM (more ...)
The composition of the MLL multiprotein complex in K562 cells was further confirmed by immunoprecipitation analysis of nuclear extracts. ASH2L1 and -2, WDR5, RBBP5, menin, and HCF-2 were each detected by specific antisera on Western blot analysis of the immunoprecipitates generated with anti-MLLN
antibodies (Fig. ). HCF-1 (a homolog of HCF-2) was also detected in the MLL immunoprecipitates (Fig. ). Multiple HCF-1 bands on Western blot analysis reflect its known proteolytic processing into several different-sized polypeptides (58
). Its coprecipitation efficiency was much less than that of HCF-2, suggesting that although MLL associates with HCF-1, HCF-2 is a preferred component of the MLL complex in K562 cells. In contrast to a previous report (44
), we were unable to coprecipitate either the corepressor Sin3A or chromatin remodeling protein BRM with MLLN
. Immunoprecipitation analysis was also performed with antibodies specific for individual components of the MLL complex, which clearly demonstrated the presence of MLLC
in immunoprecipitates of ASH2L, WDR5, HCF-1, HCF-2, and menin, respectively (Fig. ). However, MLLC
was not detected in the immunoprecipitates of Sin3A or hSNF2H, previously reported as components of an MLL supercomplex (44
FIG. 2. Coprecipitation of MLL with associated factors. (A) A nuclear extract of K562 cells (lane 1 input) was subjected to immunoprecipitation (IP) using antibodies specific for MLLN (lane 4) or MLLC (lane 5). As negative controls, precipitations were also performed (more ...)
Further evidence in support of the observed MLL complex composition was obtained by gel filtration chromatography. The fraction eluted at 500 mM KCl from heparin Sepharose (Hep500) purified according to the scheme depicted in Fig. was subjected to Superose 6 gel filtration chromatography. Fractions were concentrated by acetone precipitation and then were immunoblotted with antibodies specific for components of the MLL complex. Elution of MLLC and other complex components overlapped extensively and was broadly distributed from the void to fraction 26, peaking at fraction 24 (approximately 1 MDa), consistent with the predicted size of the MLL complex. The elution profile of menin was broader, indicating that not all menin forms a complex with MLL (Fig. ). A minor MLL peak at fraction 16 suggested the presence of another form of MLL sizing at more than 2 MDa, which is reminiscent of the MLL supercomplex (Fig. ). However, immunoprecipitates from either nuclear extract or the Hep500 fraction did not contain MLL supercomplex components, such as Sin3A or BRM (Fig. and unpublished data). In conclusion, our data demonstrate that MLL forms a multiprotein complex with ASH2L, WDR5, RBBP5, menin, and HCF proteins. We hereafter refer to this complex as MLL/HCF to distinguish it from other possible forms of MLL.
The composition of MLL/HCF shares considerable similarity with the previously characterized SET1 HMT complexes of Saccharomyces cerevisiae
and humans (41
) (Fig. ). All three complexes contain homologs of Drosophila
Ash2, a trxG
gene product required for imaginal disk pattern formation (36
). The human homolog, ASH2L, is expressed as two isoforms (denoted 1 and 2) by alternative splicing (57
). In yeast, two gene products (Bre2 and Spp1) together appear to constitute a bipartite functional homolog of Ash2 as described previously (43
). All three complexes also contain highly similar WD repeat-containing proteins. WDR5 is a mammalian homolog of two proteins (Swd2 or Swd3) which are components of the yeast SET1 complex and are required for its histone methylation (43
). RBBP5, another WD repeat-containing protein, also has a homolog (Swd1) in the yeast SET1 complex but has not yet been reported in mammalian SET1. Conversely, S. cerevisiae
lacks a homolog of HCF-1, which is a common component of the mammalian SET1 and MLL complexes (61
). It was originally identified as a host cell factor targeted by the herpes simplex virus VP16 protein (33
). Menin, a tumor suppressor protein with multiple known roles and interactions (11
), has not been reported to be present in the human SET1 complex and thus appears to be a unique component of MLL/HCF. Our data indicate that MLL forms a multimember complex whose composition is conserved in part with the SET1 complexes associated with histone H3 lysine 4 methylation, suggesting that they all employ similar enzymatic mechanisms.
FIG. 3. MLL complex composition is conserved with yeast and human SET1 methyltransferase complexes. Similarities between components of the yeast SET1, human SET1/HCF-1, and human MLL/HCF complexes are shown. The protein components of each complex are shown schematically (more ...) The SET domain of MLLC assembles methyltransferase-associated cofactors.
To define heterologous protein interactions within the MLL/HCF complex, various mutant forms of MLL were transiently expressed in 293 cells and were assessed for their ability to interact with and coprecipitate endogenous members of the complex. Under these conditions, endogenous HCF1, HCF2, ASH2L, RBBP5, and WDR5 readily coprecipitated with transfected wild-type MLL as assessed by immunoprecipitation-Western blot analysis (Fig. ). A mutant MLL lacking C-terminal sequences spanning the SET domain (Δ3820), however, lost the ability to interact and coprecipitate with ASH2L, WDR5, and RBBP5 but retained an ability to coprecipitate HCF proteins. Therefore, ASH2L, WDR5, and RBBP5 interact with the p180 MLLC
subunit through its SET domain, which is highly conserved with the mammalian and yeast SET1 proteins (4
). The homologous proteins Bre2, Swd1, Swd2, and Swd3 in the yeast SET1 complex are necessary for its full HMT activity in vivo (43
). Hence, it is likely that ASH2L, WDR5, and RBBP5 form an MLLC
subcomplex to effect HMT catalytic activity (Fig. ).
FIG. 4. Conserved methyltransferase components associate with MLLC. (A) 293 cells were transiently transfected with expression vectors encoding various MLL deletion mutants (shown schematically) containing HIS and FLAG epitope tags at their N termini. Nuclear (more ...) Conserved motif in MLLN mediates interactions with HCF proteins.
More refined mutation analysis indicated that interactions with HCF-1 and -2 required amino acids 1799 to 1802 of MLL, whereas interactions with ASH2L and WDR5 did not (Fig. ). This sequence fits a consensus (D/EHXY) for the previously defined HBM (Fig. ) found in all proteins known to physically associate with HCF-1 through its amino-terminal Kelch domain, including VP16 (18
). In addition to MLL and its paralogs, HBMs are conserved in mammalian MLL-related proteins MLL2 and SET1 but not in ALR (Fig. ). However, characterization of a menin-MLL2 complex didn't detect HCF-1 or HCF-2 (27
). Therefore, the possibility of MLL2 interaction with HCFs [signified by (+) in Fig. ] remains to be investigated. Previous studies have shown that the HCF-1 Kelch domain binds human SET1 and that the amino-terminal portion of HCF-1 (HCF-1N
) tethers SET1 with the corepressor Sin3A (61
). When Flag-tagged HCF-1 mutants were stably expressed in HeLa cells, immunoprecipitation-Western blot analysis showed that the Kelch domain of HCF-1N
was sufficient for interaction with MLL (Fig. ), whereas interaction with Sin3A was dependent on the adjacent basic region (Fig. ) as previously reported (61
). Therefore, MLL interacts with HCF proteins through its HBM, consistent with the proposed mechanism of Kelch-HBM interaction.
FIG. 5. Conserved binding motif in MLLN mediates interactions with HCF-1 and HCF-2. (A and B) 293 cells were transiently transfected with expression vectors encoding wild-type MLL or a mutant lacking the HBM (shown schematically in panel A). Nuclear extracts (more ...) Menin interacts with wild-type MLL and leukemic MLL fusion proteins.
Domain mapping revealed that menin interaction occurs within the first 1,406 amino acids of MLLN (Fig. ). This portion is consistently retained in all MLL fusion proteins associated with human leukemias. Immunoprecipitation-Western blot analysis showed that endogenous menin coprecipitated with the MLL-p300 fusion protein in transfected 293 cells (Fig. ). To further confirm that menin interacts with MLL fusion proteins, coimmunoprecipitation assays were performed with the HB cell line, which expresses endogenous MLL-ENL fusion protein due to chromosomal translocation. Immunoprecipitation of menin resulted in the coprecipitation of both wild-type and fusion MLL proteins from HB cells (Fig. , lane 6). A similar analysis using REH cells, which lack an MLL chromosomal translocation, resulted in coprecipitation of only wild-type MLL with menin. Therefore, it is likely that all MLL fusion proteins interact with menin, which is the only identified component of the MLL/HCF complex that associates with mutant MLL proteins and thus is potentially implicated in leukemia pathogenesis.
FIG. 6. Menin interacts with wild-type and oncogenic MLL proteins. (A and B) 293 cells were transiently transfected with expression vectors encoding various MLL deletion or fusion mutants (shown schematically in panel A) containing HIS and FLAG epitope tags at (more ...) Menin, but not other components, is required for maintenance of HoxA9 gene expression.
The requirement for each component of the MLL/HCF complex in the maintenance of Hox gene expression was investigated in HeLa cells. The expression of complex components was specifically eliminated by siRNA techniques, and knockdown efficiencies were estimated by Western blotting, which revealed more than 80% reduction in expression for each component (Fig. ). Quantitative real-time RT-PCR analysis showed that knockdown of MLL expression resulted in a significant downregulation (50% decrease) in HoxA9 mRNA levels relative to that of GAPDH, whereas knockdown of the control GL2 protein showed no effect on HoxA9 (Fig. ). Despite more than 80% knockdown efficiency of ASH2L, WDR5, HCF-1, and HCF-2, their reductions had no measurable effect on HoxA9 expression, suggesting that low levels of these components may be sufficient for maintenance of HoxA9 transcription in HeLa cells. Conversely, menin knockdown caused downregulation of HoxA9 transcripts comparable to that observed for MLL knockdown (Fig. ). Therefore, we conclude that menin is an essential component that is required for maintenance of HoxA9 gene expression.
FIG. 7. Menin is required for maintenance of Hox gene expression. (A and B) HeLa cells were subjected to three rounds of transfection with siRNAs specific for transcripts encoding components of MLL/HCF complex (indicated at the tops of gel lanes). RNA and protein (more ...)