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, 42 (11), 6901-20

The Interaction of MYC With the Trithorax Protein ASH2L Promotes Gene Transcription by Regulating H3K27 Modification

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The Interaction of MYC With the Trithorax Protein ASH2L Promotes Gene Transcription by Regulating H3K27 Modification

Andrea Ullius et al. Nucleic Acids Res.

Abstract

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.

Figures

Figure 1.
Figure 1.
ASH2L interacts with MYC in vitro and in cells. (A) MYC was immunoprecipitated from U2OS cells using the mAb 4H3. HA-tag-specific antibodies served as control. The co-immunoprecipitation of ASH2L was analyzed by immunoblotting using the ASH2L-specific 4C5 mAb. MYC was detected using pAb N262. The different lanes were run on the same western blot. (B) ASH2L was immunoprecipitated from lysates of Jurkat T cells using the mAb 4C5. ASH2L was detected on western blots with pAb 548 and the co-immunoprecipitated MYC with MYC-specific N262 polyclonal antibodies. Antibodies specific for the HA-tag were used as negative control. (C) In situ PLA in U2OS cells using primary mAb 4C5 to detect ASH2L and primary N262 purified pAb to detect MYC and species-specific secondary antibodies with oligos attached to them (PLA probes). For negative control, one primary antibody was assayed with the species-specific secondary antibody. The number of foci were counted from 50 cells of three independent experiments, displayed as mean value and standard deviation (using students’ t-test). The inset shows two representative cells with foci in blue and the DNA stained in green. (D) GST-pull-down assays were carried out with different fragments of MYC (as indicated) fused to GST and GST alone as control. The binding of in vitro transcribed and translated, 35S-methionine-labeled ASH2L was analyzed by SDS-PAGE and autoradiography. The fusion proteins used are shown below in a Coomassie-Blue-stained gel (CB). (E) GST-pull-down assay of bacterially expressed and purified MYC and ASH2L fusion proteins were performed with GST-MYC-C176 containing the ASH2L interaction domain and with His6-ASH2L-N387 containing the N-terminal 387 amino acids that are sufficient for the interaction with MYC. (F) Summary of the interactions of ASH2L with MYC of in vitro pull-down assays and of co-immunoprecipitation experiments obtained from HEK293 cells upon transient expression of the respective fragments. (G) GST-pull-down assays were carried out with different fragments of ASH2L (as indicated) fused to GST and GST alone as control. The binding of in vitro transcribed and translated, 35S-methionine-labeled MYC was analyzed by SDS-PAGE and autoradiography. The bottom panel shows the schematic organization of ASH2L. PHD, atypical plant homeodomain; HWH, helix-winged-helix domain; NLS, nuclear localization signal; SPRY, an SP1a and RYanodine receptor domain; SDI, SDC1/DPY30 interaction motif (54,114–116). The results of the GST-pull-down assays are summarized schematically at the bottom.
Figure 2.
Figure 2.
Recruitment of H3K4-specific methyltransferase activity by MYC is ASH2L dependent. (A) ASH2L was immunoprecipitated from Jurkat T cell F-buffer lysates with the indicated pre-immune and immune serum (548) and mAbs (4B5 and 4C5) and an isotype-specific control (5F4). The ASH2L-associated MTase activity was measured on core histones with the use of radiolabeled 3H-S-adenosylmethionine. The CB-stained gel shows the input of core histones. (B) ASH2L was immunoprecipitated from lysates of different cells using the ASH2L-specific 548 polyclonal antiserum (i) or the corresponding pre-immune serum (pi) or a combination of ASH2L-specific mAbs 4B5 and 4C5, and 5F4 for control. The ASH2L-associated MTase activity was measured on core histones with the use of radiolabeled 3H-S-adenosylmethionine. (C) The specificity of ASH2L-associated MTase activity from HEK293 was analyzed on recombinant histone H3 by using K4 and K9 methylation-specific antibodies as indicated. (D) ASH2L was immunoprecipitated from Jurkat T cell lysates. The indicated recombinant GST- or MBP-fusion proteins or GST and MBP alone as control were tested as substrates for ASH2L-associated MTase activity using 3H-S-adenosylmethionine. The arrowheads in the CB-stained gel indicate the input of the respective fusion protein. (E) and (F) Control vector, Flag-tagged MYC wt and mutants were expressed transiently in HEK293 cells, immunoprecipitated from F-buffer lysates using a Flag-specific antibody and the MYC-associated MTase activity was measured on recombinant GST-H3 N-terminal tails using 3H-S-adenosylmethionine. (G) HEK293T cells were transiently transfected with Flag-tagged MYC and pSuper constructs expressing an ASH2L-specific shRNA or a control shRNA. After immunoprecipitation of MYC from F-buffer lysates using a Flag-specific antibody, MYC-associated MTase activity was measured on recombinant Histone H3 using an H3K4me3-specific antibody. The expression of relevant proteins are shown for control. (H) HeLa cells were transiently transfected with pSuper constructs expressing either an ASH2L-specific shRNA or a control shRNA. The presence of ASH2L (mAb 4C5) and H3K4me3 (Abcam 8580) was visualized by immunofluorescence. Arrowheads and arrows indicate transfected and untransfected cells, respectively.
Figure 3.
Figure 3.
MYC and ASH2L interact at promoters. (A)–(D) P493-6 B cells were growth-arrested by the addition of tetracycline, which represses MYC expression, for 72 h. ChIP was performed six hours after either with (+MYC) or without removal of tetracycline (-MYC) using specific antibodies against MYC (A), ASH2L (B), WDR5 (C), or RbBP5 (D). Immunoprecipitated DNA was amplified by quantitative PCR (qPCR) with primers for CCND2, ODC and NCL promoter regions and a control region 22 kbp upstream of the CCND2 promoter (ctrl), as indicated in the scheme. The E-boxes (red, known binding sites for MYC) in ODC and NCL are promoter proximal, the two E-boxes in CCND2 are 1.2 kbp upstream of the transcriptional start site. (E) and (F) P493-6 B cells were treated as described for panel A. Sequential ChIP was carried out by first immunoprecipitating either ASH2L- (E) or MYC-containing (F) complexes, followed by a second ChIP with antibodies against MYC, ASH2L, CBP/p300, or IgG control. DNA fragments were amplified by qPCR as indicated for panel A. Error bars represent s.d. of PCR triplicates.
Figure 4.
Figure 4.
MYC stimulates histone H3 acetylation and inhibits H3K27 trimethylation but does not modulate H3K4 trimethylation. (A)–(F) P493-6 B cells were treated as described in the legend to Figure 3. ChIP was performed with antibodies against H3K4me3 (A), H3ac (B), H3K9ac (C), H3K27ac (D), H3K27me3 (E) and CBP/p300 (F). (G) Sequential ChIP was carried out by first immunoprecipitating MYC-containing complexes, followed by a second ChIP with antibodies against H3ac, H3K9ac, H3K27ac, H3K4me3, H3K27me3, or IgG control. The DNA fragments were amplified by qPCR with the primers indicated in the scheme. Error bars represent s.d. of PCR triplicates.
Figure 5.
Figure 5.
ASH2L is required for efficient gene expression and H3K4 trimethylation. (A) HEK293T cells were transiently transfected with siRNA oligo pools targeting ASH2L or MYC mRNA or a control oligo pool (Ctrl). Expression of ASH2L, MYC, CCND2, ODC and NCL mRNA was analyzed by quantitative RT-PCR and normalized to GUS. Error bars represent s.d. (n = 4). (B) Cell lysates of HEK293T cells from an experiment shown in panel A were used for western blot analyses with the indicated antibodies and actin as loading control. (C)–(H) HEK293T cells were transiently transfected with siRNA pools targeting ASH2L mRNA. Subsequent ChIP experiments were performed with antibodies against ASH2L (C), H3K4me3 (D), RbBP5 (E), WDR5 (F), MYC (G) and H3ac (H). DNA fragments were analyzed as described in the figures before. Error bars represent s.d. of PCR triplicates.
Figure 6.
Figure 6.
ASH2L and MYC control modification of H3K27. (A)–(F) HEK293T cells were transiently transfected with siRNA oligo pools targeting MYC mRNA. ChIP assays were performed with antibodies against MYC (A), ASH2L (B), H3K4me3 (C), H3K27ac (D), H3K27me3 (E) and CBP/p300 (F). DNA fragments were analyzed as described in the figures before. (G) and (H) HEK293T cells were transiently transfected with siASH2L oligo pools and used in ChIP assays with antibodies against H3K27ac (G) and H3K27me3 (H). The DNA fragments were amplified by qPCR with the primers indicated in the scheme. Error bars represent s.d. of PCR triplicates.
Figure 7.
Figure 7.
Binding of MYC and WDR5, a core subunit of ASH2L–KMT2 complexes, correlates with gene expression. (A) Proportion of ChIP-Seq peaks of WDR5, MYC and both MYC and WDR5 in comparison to distinct types of regulatory regions in NHEK cells. Chromatin regions were defined by the presence of ChIP-Seq peaks of distinct histone modifications or combinations thereof and the distance of the regions to transcriptional start sites. (B) The proportion of MYC (Jaspar Motif MA0147.1) and ASH2L (54) binding sites inside WDR5, MYC or MYC+WDR5 ChIP-Seq peaks. In all cases, the number of MYC and ASH2L-binding sites were significantly higher than binding sites in randomly selected genomic regions (*indicate conditions with a z-test p-value < 0.0001). (C) The log of the number of RNA reads per kilobase pairs on genomic regions close (+/- 500 bp) to WDR5, MYC and MYC+WDR5 ChIP-Seq peaks. We only considered those ChIP-Seq peaks overlapping with the 200 bp upstream regions of transcriptional start sites. (D) RNA-Seq-derived strand specific expression (red) and ChIP-Seq profiles (blue) and their corresponding peaks (green) of genomic regions around ODC, NCL and CCND2.
Figure 8.
Figure 8.
Schematic and simplified depiction of the interaction of MYC with ASH2L–KMT2 complexes and their effects on core histone modifications and gene transcription. In the repressed state, characterized by H3K27me3, no ASH2L–KMT2 complexes are bound to chromatin. In open chromatin, specific promoters are associated with ASH2L–KMT2 complexes resulting in high H3K4me3. Two forms of open chromatin are indicated, one is characterized by H3K27me3 in addition to H3K4me3. This is referred to as bivalent chromatin and promoters with these two marks are typically poised. How ASH2L–KMT2 complexes are recruited initially is not well understood. This may occur through direct interaction of the KMT2 complex with chromatin or through an unknown transcription factor (referred to as X). KDM6 enzymes, which demethylate H3K27, and CBP/p300, which acetylate H3K27, have been reported to interact with KMT2 complexes and may therefore be located on promoters prior to MYC binding. In the absence of MYC, H3K27 modifying enzymes seem to be largely inactive. Promoters switch to a transcriptionally active mode upon binding of MYC, which we postulate to result in the activation of these H3K27 modifying enzymes (indicated by the color change). This leads to a reduction of H3K27me3 and concomitant increase in H3K27ac, which combined with H3K4me3 marks open chromatin with active gene transcription.

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