The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification

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.

transcribed/translated material was loaded as input control. The respective input of the GST proteins is shown by the Coomassie Blue (CB) stained gel. (B) Binding of recombinant His 6 -WDR5 to  and GST alone were analyzed by GST-pull-down experiments and immunoblotting of WDR5, respectively. Onetenth of His 6 -WDR5 was loaded as input control. The respective input of GST proteins is shown by Ponceau staining. (C) HEK293T cells were transfected with MYC and Flag-tagged MLL2 fragment (653 aa of C-terminus), WDR5, RbBP5, or ASH2L. Cells were harvested 48 hours after transfection and lysed in co-IP buffer. MYC was immunoprecipitated with MYC-specific antibodies (N262) and precipitates were analyzed by Western blotting with α-Flag antibodies. Whole cell lysates were subjected to Western blot analysis using Flag-and MYC-specific (N262) antibodies. The arrow head in the upper panel indicates RbBP5.

Supplementary Figure S4
Characterization of P493-6 B cells. P493-6 cells were treated with 0.1 µg/ml tetracycline for 72 hours. Removal of tetracycline was achieved by washing the cells with PBS and adding fresh RPMI medium containing 10% FCS. Cells were harvested after the addition of fresh medium without tetracycline at the indicated time points. For control cells were grown in the absence of tetracycline and analyzed in parallel (Ø). (A) Expression of MYC mRNA was analyzed by quantitative RT-PCR and normalized to the expression of GUS. The relative expression of the normalized MYC mRNA is shown with the value set to 1 at the 0 hour time point. Whole cell lysates of P493-6 cells were prepared 0, 3, and 6 hours after tetracycline removal and MYC protein levels were measured by Western Blot analysis using MYC-specific antibodies (N262) and actin as loading control (inset). (B) P493-6 were fixed with methanol and stained with propidium iodide. Distribution of cells in G1, S or G2/M phase was determined using flow cytometry analysis (FACS). (C) Expression of CCND2, ODC, and NCL mRNA was analyzed at the different time points by quantitative RT-PCR and normalized to GUS. The relative expression of the normalized CCND2, ODC, and NCL mRNAs is shown with the values set to 1 at the 0 hour time point.

Supplementary Figure S5
(A) P493-6 B cells were treated with 0.1 µg/ml tetracycline for 72 hours. ChIP assays were performed either directly (-MYC) or six hours after tetracycline removal (+MYC) with IgG control antibodies. 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. (B) HEK293T cells were transiently transfected with siRNA oligo pools targeting ASH2L mRNA or with a control oligo pool (Ctrl). ChIP assays were carried out with IgG control antibodies. Purified DNA was analyzed by qPCR as described in A. (C) HEK293T cells were transiently transfected with siRNA oligo pools targeting MYC mRNA or with a control oligo pool (Ctrl). ChIP assays were performed with IgG control antibodies. DNA fragments were analyzed as described in A.

Supplementary Figure S6
ChIP assays of HEK293T cells, transfected with single siRNA oligos (#1 in panels A, C, E, and G or #2 in panels B, D, F, and H) targeting ASH2L mRNA or with a control siRNA oligos (Ctrl), were performed with antibodies against ASH2L (A+B), H3K27ac (C+D), H3K27me3 (E+F), and IgG as control (G+H). 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.

Supplementary Figure S7
ChIP assays of HEK293T cells, transfected with single siRNA oligos (#1 in panels A, C, E, and G or #2 in panels B, D, F, and H) targeting MYC mRNA or with a control siRNA oligos (Ctrl), were performed with antibodies against MYC (A+B), H3K27ac (C+D), H3K27me3 (E+F), and IgG as control (G+H). 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.  Figure S1 revised