Fig. 3

Gain of function of VHL mutations impact genome-wide HIF2α chromatin binding sites in 786-O cells. A, B Western blot determination of protein expression levels in 786-O cells stably expressing VHL WT and mutants, with or without treatment of the proteasome inhibitor MG132. C Genomic annotation of HIF2α ChIP-seq peaks from VHL WT mutant expressing cells. Genomic features of binding peaks were visualized in pie charts, where the demonstrated genomic features, including promoter regions within 1 kb, 1–2 kb and 2–3 kb; gene body (5′UTR, 3′UTR, exons, and introns); downstream elements, and distal intergenic regions. UTR, untranslated region. D Visualization of a HIF2α binding site at the promoter of a known hypoxia-regulated gene, VEGFA. E The top enriched motifs in the HIF2α ChIP-seq peaks determined by HOMER software. F, G Heatmap indication of HIF2α chromatin binding intensity based on ChIP-seq reads in 786-O cells expressing VHL WT vs. S65P mutant (F) or VHL WT vs. S65W mutant (G). Signals within 3 kb around ChIP-seq peak center are demonstrated in a descending order for each clustered HIF2α binding event (common or unique). Plots in the right panels of F or G display average signal of HIF2α binding at the indicated clustered regions