Fig 2: Euchromatic histone methyltransferases (EHMTs) as inversely regulated genes with CXCL10 level in high-risk neuroblastomas and its association with MYCN amplification. (A) Genes correlating with CXCL10 expression in INSS 4 NBs ranked by increasing Pearson correlation coefficients. Epigenetic modifiers from Xu et al30 are highlighted. Red: positively correlated; blue: negatively correlated. (B) As in (A) but showing genes correlating with MYCN expression. (C) Boxplots showing EHMT2 and EHMT1 expression (RNA-seq, log2) in INSS 4 NBs by MYCN status. (D) Boxplots showing EHMT2 and EHMT1 expression (3′mRNA-seq, log2) in human NB cell lines by MYCN status. (E) Western blots for MYCN, EHMT1, EHMT2, EZH1, EZH2 and β-actin in siMYCN or non-targeting non-targeting siRNA (siNT)-treated SK-N-BE and IMR-32 cells. Representative blots of biological replicates (n=4). (F, G) Quantification of experiment described in (E) from biological replicates (n=4). Error bars; SD. (H) and (I) MYCN-binding peaks in the genomic regions of EHMT2 (upper panels) and EZH2 (lower panel) by ChIP-seq. Cell lines and data set as indicated. (J) and (K) EHMT2 and EHMT1 expression in NB cells versus other cancer cells from the CCLE database. Statistics: two-sided unpaired t-tests (C, D, J, K). Two-sided unpaired t-test with logarithmic values (F, G). Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. *p<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. NB, neuroblastoma.

Fig 3: MCV LT activates EZH2 gene expression by targeting the pRb-E2F pathway. A. qRT-PCR analysis for Ezh1 and Ezh2 mRNA expression in BJ cells stably transduced with empty vector (Empty), HA-E6, HA-E7, and HA-E7LYCDK pRb binding mutant. Relative mRNA expression to empty vector control was determined by the 2-ΔΔCt method with GAPDH as a normalization control. Error bars represent standard deviation. B. EZH2, EZH1, and E2F-1 protein expression were examined by immunoblot using stable cells from Fig. 1A. Hsp70 was used as a loading control. C. qRT-PCR analysis for Ezh1 and Ezh2 mRNA expression in BJ cells stably transduced with empty vector (Empty), MCV sT, tumor-derived 339 LT, and 339LTLFCDK pRb binding mutant. Relative mRNA expression to empty vector control was determined by the 2-ΔΔCt method with 18S as a normalization control. Error bars represent standard deviation. D. BJ cells used in Fig. 1C were immunoblotted for EZH2, EZH1, and E2F-1 expression. Hsp70 was used as a loading control.

Fig 4: EZH2 is regulated by MCV T antigen oncogene in MCV(+) MCC cells. A. qRT-PCR for Ezh2, Ezh1, and T antigen mRNA in MKL-1 cells transduced with shRNAs targeting both LT and sT antigens (sh panT) or only small T antigen (sh sT) and non-targeting control (sh Ctrl). Relative expression to empty vector control was calculated by the 2-ΔΔCt method with 18S as a normalization control. Error bars represent standard deviation. B. Immunoblot showing EZH2, EZH1, E2F-1, LT, and sT protein expression in MKL-1 MCV(+)MCC cells with panT antigen and sT antigen knockdown. Hsp70 was detected as a loading control. The bottom graph shows Li-COR quantification of EZH2 protein expression from multiple independent experiments (Fig.S2D). C. Immunoblots with MCV(+) cells MKL-2, CVG-1, and MS-1 transduced with sh panT showing the effect of EZH2 reduction. Hsp70 was detected as a loading control. D. Immunoblots for EZH2, LT, and Hsp70 to examine the effect of LT knockout on EZH2 protein expression in MKL-1 and MKL-2. The top panel shows the schematic of sT and truncated LT (tLT) gene. Arrows indicate the position of two sgRNAs sg LT1240 and sg LT1435 that introduce indels before and after LxCxE motif (red box). E. Representative immunohistochemical expression of EZH2 as compared to MCPyV LT in representative cases of virus-positive (VP) and virus-negative (VN) MCC. F. Comparison of EZH2 immunohistochemical expression (quantitated by h score) in VP-MCC and VN-MCC tumors. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)