Fig. 7: EPAS1-enhancer 2 controls downstream effects of HIF-2α and tumor growth.

a Representative immunoblot (n = 3) for HIF-2α and β-actin protein in lysates from single clones of EPAS1-enhancer 2 defective (E2_HIF) or control (non-targeting, nt) 786-0 cells. b Xenograft tumor assay in NOD/SCID-gamma mice using EPAS1-enhancer 2 defective (E2_HIF ko) or intact (nt) clones of 786-0 cells. Volume values are mean ± SD from 6 tumors per cell clone. Two-way ANOVA followed by Bonferroni’s post hoc test comparing tumor volume between mean nt and E2_HIF ko at the indicated time points. Significant differences (**p < 0.01) were detected for comparisons between mean values of E2_HIF ko (2.1 or 2.2) with nt (0.1 or 0.2) xenografts for weeks 8, 9 and 10. c Weight of the explanted xenograft tumors. Data shown are median and interquartile range from 5 (E2_HIF 2.1) or 6 (nt 0.1, nt 0.2, E2_HIF2.2) tumors. One-way ANOVA followed by Bonferroni’s post hoc test. d Volcano plot from RNA-seq experiments in E2_HIF ko versus nt clones of 786-0 cells. HIF-2α mRNA as well as HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. Data is from the two clones of cells per condition from a) and b). e GSEA for HIF-binding sites in 786-0 cells and RNA-seq data (n = 2 clones) from d). DEG: differentially expressed genes, ES: enrichment score. p-value was estimated using adaptive multilevel Monte Carlo scheme and adjusted for multiple testing with the Benjamini–Hochberg method. f CUT&Tag-seq (C&T) for HIF-1β and H3K27ac as well as ATAC-seq at EPAS1-enhancer 2 and the control locus EGLN3 from E2_HIF ko versus nt clones of 786-0 cells. g CUT&Tag-seq signals for HIF-1β or H3K27ac integrated over 543 ChIP-seq pre-defined HIF1-β binding sites in E2_HIF ko versus nt clones of 786-0 cells. Non-HIF binding enhancers served as control. Signals were centered on the H3K27ac peak within the enhancers. h Comparison of log₂ (fold change) expression values of genes significantly regulated in E2_HIF ko 786-0 cells (n = 2 clones) or through HIF-2 inhibition by PT2385 in 786-0 cells⁴⁶. HIF-target transcripts NDRG1, VEGFA, and CCND1 are indicated. i The transcriptional circuitry of HIF-2α expression at the EPAS1 gene locus. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560.