Fig. 1: Identification of EZH2 as a MYCN/MYC common binding partner.

a, b Volcano plots showing interacting proteins of MYCN (a) and MYC (b). Associated proteins were immunoprecipitated from neuroblastoma Kelly cells (a) and small cell lung carcinoma NCI-H2171 cells (b) using specific MYCN and MYC antibodies, respectively. Proteins significantly enriched from three independent experiments with a fold-change >2 and p value < 0.05 against the IgG group are displayed as red dots. c Venn diagram of MYCN- and MYC-interacting partners. d Mass spectrometry results of MYCN and MYC common binding partners. The axes show the fold-change of α-MYCN/MYC-associated proteins relative to α-IgG control. e Co-immunoprecipitation (Co-IP) to detect interaction between endogenous MYCN and PRC2 components. Lysates of Kelly cells were treated with a DNase/RNase (10 μg/mL) combination and subjected to co-IP for detecting protein interaction. The asterisk denotes a nonspecific band. f GST pull-down to identify the direct MYCN-binding partner. Recombinant His-tagged MYCN was respectively incubated with GST-tagged EZH2, SUZ12, and EED in vitro before pulldown assay with GST beads, and MYCN interaction was analyzed by immunoblot with anti-His antibody. The asterisk denotes a nonspecific band. g Schematic representation of MYC, MYCN, and EZH2. MB1, 2, and 3: MYC box1, 2, and 3, respectively; bHLHZip: helix–loop–helix domain; DNMT BR: DNA methyltransferase-binding region; CDYL BR: chromodomain Y-like protein-binding region; CXC: cysteine-rich domain, SET: Su(var)3–9, enhancer of zeste, trithorax domain. h Characterization of the MB motif required for MYCN and EZH2 interaction. 293T cells were transfected with plasmids expressing epitope-tagged EZH2, MYCN, or its MYC box deletion (ΔMB) mutants as shown. Anti-Flag-associated precipitates were used for detection of EZH2 binding. i Analysis of EZH2 CDYL-BR and MYCN/MYC MB2 interaction. 293T cells were transfected with plasmids expressing EZH2-CDYL-BR, and Streptavidin-binding peptide (SBP)-tagged MYC or MYCN for 48 h as shown. SBP pull-down was performed to analyze the protein interaction as indicated. The experiments were independently repeated three times with similar results (e, f, h–i). Source data are provided as a Source data file.