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. 2015 Jul 2;35(15):2658–2672. doi: 10.1128/MCB.00161-15

FIG 6.

FIG 6

A HIF2α-specific inhibitor blocks the dissociation of nucleosome structures in the EPO gene under hypoxic conditions in human hepatocytes. (A) Immunoblots for HIF1α and HIF2α in Hep3B human hepatoma cells under normoxic (N) and hypoxic (H24 [1% oxygen for 24 h]) conditions. α-Tubulin was used as an internal control. Both HIF1α and HIF2α proteins accumulated in cells treated with a PHD inhibitor (FG4592) or in cells under hypoxic stimulation, and the protein levels were not altered by supplementation with HIF inhibitors (HIF2 antagonist 2 and acriflavine) under hypoxic conditions. (B) RT-qPCR of CA9, PGK1, and EPO mRNA expression in Hep3B cells cultured with vehicle (v) or HIF inhibitors (HIF2 antagonist 2 and acriflavine) under normoxic (N) or hypoxic (H24) conditions. β-Actin was used as an internal control for RT-qPCR. The expression levels under normal conditions with vehicle supplementation (v/N) were set as 1.0. The data are means and SD from 4 independent experiments. *, P < 0.01 compared with hypoxic samples supplemented with vehicle (v/H24). A HIF2α-specific inhibitor (HIF2 antagonist 2) strongly suppressed hypoxic induction of EPO expression but did not affect the expression of PGK1. (C) Nucleosome occupancy of chromatin loci for the CA9, PGK1, and EPO genes was detected via nuclease accessibility in Hep3B cells cultured with vehicle (v) or HIF inhibitors (HIF2 antagonist 2 and acriflavine) under normoxic (N) or hypoxic (H4 [1% oxygen for 4 h] and H24 [1% oxygen for 24 h]) conditions. The indicated loci were detected by qPCR of nuclease-digested and undigested chromatin from Hep3B cells, and the amount found in undigested samples was set as 100%. The values are means and SD from 4 independent experiments. *, P < 0.01 compared with vehicle-treated samples (v) under each condition. Nuclease accessibility was high in the PGK1 promoter region in all samples, indicating that this region is free from nucleosome structure under both normoxic and hypoxic conditions. The promoter, gene body (intron 1), and hepatic enhancer (HE) of the EPO gene and the CA9 promoter region were well protected from nuclease digestion under normal conditions (v/N), and nuclease accessibility increased under hypoxic conditions. Note that the formation of the nucleosome-free region under hypoxic conditions was repressed by HIF inhibitors.