Figure 4. MYCN directly activates EZH2 expression.

(A) Correlation of MYCN mRNA and EZH2 mRNA expression in primary neuroblastoma (NBL) tumor samples based on Affymetrix data GSE12460. P computed with the rcorr function (R-CRAN, Hmisc library). (B) EZH2 mRNA expression across Cancer Cell Line Encyclopedia cancer cell lines with neuroblastoma cell lines in red. (C) Immunoblot of protein levels of EZH2, MYCN, and MYC in MYCN-amplified compared with MYCN-nonamplified neuroblastoma cells. (D) Effect of MYCN suppression on EZH2 protein levels. Results are representative of 2 independent experiments. (E and F) Effect of conditional overexpression of MYCN on EZH2 expression at the transcription level (E) and the protein level (F) in the MYCN-nonamplified neuroblastoma cell line SHEP. The SHEP-Tet-MYCN cell line was generated by stable transduction of SHEP with a Tet-off MYCN construct. MCM7 is a known MYCN target gene. SK-N-AS is an unmodified MYCN-nonamplified neuroblastoma cell line. Mean ± SEM of 3 independent experiments shown. *P < 0.05 by 2-tailed Student’s t test. (G) ChIP–quantitative PCR with MYCN antibody showing the enrichment of MYCN in the promoter region of EZH2 in MYCN-overexpressing SHEP-Tet-MYCN cells. MCM7 is a positive control, and ZIC3 is a negative control. TSS, transcription start site. Results are representative of 2 independent experiments; mean ± SD of 3 technical replicates is shown.