Fig. 5: Lineage-specific transcription factors govern activity of EPAS1-enhancer E2.
From: HIF sustain a transcriptional regulatory circuit of EPAS1 expression in renal clear cell carcinoma
a ChIP-seq tracks for HIF-1β, PAX825, H3K27ac and HNF-1β CUT&Tag-seq track from 786-0 cells at EPAS1-enhancer E2. Binding sites for HNF-1β (E2_HNF-1β) and PAX8 (E2_PAX8) within KIRC_10767 are highlighted in blue. HIF-binding site is marked in orange. b Expression qPCR analyses for HIF-2α mRNA in lysates from 786-0 cells depleted for the indicated transcription factor (HNF-1β or PAX8). Values are mean + SD from 5 (HNF-1β) or 4 (PAX8) independent experiments. Statistical significance was assessed by a two-tailed one sample t-test with a hypothetical value of 1. c Immunoblot analyses for HIF-2α, HNF-1β, and β-actin in lysates from 786-0 cells depleted for HNF-1β using CRISPR/Cas9. Representative blot from two independent experiments with similar results. d Immunoblot analyses for HIF-2α, PAX8, and β-actin in lysates from 786-0 cells depleted for PAX8 using CRISPR/Cas9. Representative blot from two independent experiments with similar results. e ATAC-seq and CUT&Tag-seq tracks of the chromatin activity marker H3K27ac from single clones of HNF-1β knock-out, PAX8 knock-out or control (nt) clones of 786-0 cells at EPAS1-enhancer E2. The ATAC KIRC element 10767 harboring binding sites for HNF-1β and PAX8 is highlighted in blue, the HIF-1β binding site at KIRC_10770 is marked in orange. f Schematic depiction of transcription factor binding sites for HNF-1β, PAX8 and HIF-1β at EPAS1-enhancer E2. Positions of sgRNAs used for knock-out of the respective binding sites are indicated in orange. Created in BioRender. Naas, S. (2025) https://BioRender.com/w24j560. g Expression qPCR analysis for HIF-2α mRNA in pools of 786-0 cells transfected with sgRNA targeting the HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) binding site at the EPAS1-enhancer E2. Cells treated with non-targeting sgRNA served as controls. Values are mean + SD. p-values were determined by a two tailed one sample t-test with a hypothetical value of 1. n = 5 (E2_HNF-1β) or n = 6 (E2_PAX8) independent experiments. h Immunoblot analyses for HIF-2α and β-actin in 786-0 cells with intact (nt) or mutated binding sites for HNF-1β (E2_HNF-1β) or PAX8 (E2_PAX8) at the EPAS1-enhancer E2. Representative blot from two independent experiments with similar results.
