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

Ullius A, Lüscher-Firzlaff J, Costa IG, Walsemann G, Forst AH, Gusmao EG, Kapelle K, Kleine H, Kremmer E, Vervoorts J, Lüscher B - Nucleic Acids Res. (2014)

Bottom Line: We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin.MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, 52074 Aachen, Germany.

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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.
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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.

Mentions: In a previous study, we identified three proteins that interact with MYC (34). One of these proteins is ARTD10/PARP10, a mono-ADP-ribosyltransferase (57). The other two proteins were identified as ASH2L and nucleolin. ASH2L was of interest to us because it has been described as a component of KMT2 group MTase complexes (14,58,59). Moreover, we found that ASH2L possesses transforming activity together with Ha-RAS in rat embryo fibroblasts and that the ASH2L protein, but not the mRNA, is overexpressed in the majority of human tumors (38,60). In the following, we have studied the interaction of MYC with ASH2L. To verify our initial purification result, MYC was immunoprecipitated (IPed) from lysates of U2OS cells using MYC-specific mAbs. We detected ASH2L in these but not in the control immunoprecipitates (IPs) (Figure 1A) with mAbs that we generated and that detect ASH2L specifically (Supplementary Figure S1A and B). These MYC complexes also contained nucleolin (data not shown) and an interaction between ASH2L and nucleolin was also seen (Supplementary Figure S1C). Moreover, ASH2L was co-IPed from Jurkat T cells and HEK293 cells using MYC- and MAX-specific antibodies, MAX being the heterodimeric partner of MYC (Supplementary Figure 1C and D). In a reciprocal experiment, ASH2L was IPed and MYC was detected in the complex (Figure 1B). Together these findings suggest that MYC interacts with ASH2L in cell extracts. To address whether MYC and ASH2L are also in close proximity in cells, we performed PLAs (45,61). MYC- and ASH2L-containing structures were identified exclusively in cell nuclei (Figure 1C), further supporting an interaction of ASH2L with MYC.


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

Ullius A, Lüscher-Firzlaff J, Costa IG, Walsemann G, Forst AH, Gusmao EG, Kapelle K, Kleine H, Kremmer E, Vervoorts J, Lüscher B - Nucleic Acids Res. (2014)

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.
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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.
Mentions: In a previous study, we identified three proteins that interact with MYC (34). One of these proteins is ARTD10/PARP10, a mono-ADP-ribosyltransferase (57). The other two proteins were identified as ASH2L and nucleolin. ASH2L was of interest to us because it has been described as a component of KMT2 group MTase complexes (14,58,59). Moreover, we found that ASH2L possesses transforming activity together with Ha-RAS in rat embryo fibroblasts and that the ASH2L protein, but not the mRNA, is overexpressed in the majority of human tumors (38,60). In the following, we have studied the interaction of MYC with ASH2L. To verify our initial purification result, MYC was immunoprecipitated (IPed) from lysates of U2OS cells using MYC-specific mAbs. We detected ASH2L in these but not in the control immunoprecipitates (IPs) (Figure 1A) with mAbs that we generated and that detect ASH2L specifically (Supplementary Figure S1A and B). These MYC complexes also contained nucleolin (data not shown) and an interaction between ASH2L and nucleolin was also seen (Supplementary Figure S1C). Moreover, ASH2L was co-IPed from Jurkat T cells and HEK293 cells using MYC- and MAX-specific antibodies, MAX being the heterodimeric partner of MYC (Supplementary Figure 1C and D). In a reciprocal experiment, ASH2L was IPed and MYC was detected in the complex (Figure 1B). Together these findings suggest that MYC interacts with ASH2L in cell extracts. To address whether MYC and ASH2L are also in close proximity in cells, we performed PLAs (45,61). MYC- and ASH2L-containing structures were identified exclusively in cell nuclei (Figure 1C), further supporting an interaction of ASH2L with MYC.

Bottom Line: We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin.MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, 52074 Aachen, Germany.

Show MeSH