Fig. 4

EZH2 transcriptionally upregulates IDH2 to promote metabolic rewiring

(A) Diagram of TCA cycle. TCA cycle related genes in Fig. 3I are indicated in orange color

(B) qRT-PCR analysis of TCA cycle related genes in OVCAR8 and OVCAR4 cells upon 5µM DZNep or GSK126 treatment for 72 h. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

(C-D) qRT-PCR analysis of EZH2 and IDH2 levels upon Dox-induced EZH2 silencing in indicated cells. IDH2 level was concomitantly reduced upon Dox-induced EZH2 silencing (C), and ectopic expression of EZH2 or EZH2-ΔSET restored the level of IDH2 (D). Data are presented as mean ± SD. *P < 0.05, **P < 0.001, ***P < 0.001

(E-F) Dual-luciferase reporter assay of IDH2 promoter activity in OC cell lines upon pharmacological or genetic inhibition of EZH2. The OVCAR8 and OVCAR4 cells were treated with 5µM DZNep or GSK126 for 48 h before luciferase reporter assay (E). The indicated cells were treated with 1 µg/mL Dox for 48 h before luciferase reporter assay (F). Data are presented as mean ± SD. **P < 0.01, ***P < 0.001

(G) ChIP assay in OVCAR8 cells. A specific antibody against EZH2 but not isotype IgG, SUZ12, or H3K27me3 could capture the fragment containing EZH2 binding site in the IDH2 promoter region, which was amplified by specific primers (top) by qRT-PCR (bottom)

(H) ChIP-seq profiling showed the ChIP-seq signal for EZH2, H3K4me3, H3K27me3 and SUZ12 at the genomic loci of IDH2 in OVCAR8 cells. ChIP-seq profiling also showed the ChIP-seq signal for EZH2 and H3K27me3 in two MLL1-rearranged leukaemia cell lines in GSE180448.

(I-J) OCR assays upon pharmacological (I) or genetic (J) inhibition of EZH2. Representative OCR pattern as a function of time (in min), normalized to total protein levels. Cells were pretreated with 5 µM DZNep, GSK126 or 1 µg/mL doxycycline for 72 h before OCR measurement. Data are shown as mean ± SD of three replicates per treatment