Fig. 1.

Hypoxia rewires the GR cistrome. (A) HeLa cells were transfected with 2 μg TAT3-luc, cultured under normoxic conditions in the presence or absence of the hypoxia mimetic deferoxamine (100 μM), or in an anoxic chamber then treated with dexamethasone (Dex) for 16 h prior to lysis and luciferase assay. (B) Cells cultured in normoxia, anoxia, treated with deferoxamine or transiently transfected with HIF-1α were lysed and immunoblotted for HIF-1α or tubulin as a loading control. (C) Cells treated with deferoxamine were incubated with or without 100 nM dexamethasone for 1 h, lysed, immunoprecipitated for GR, and then immunoblotted for HIF-1α. (D) HeLa cells were cultured in either normoxia or hypoxia overnight, and GR cistrome identified by ChIP-seq. The heatmap shows binding peak intensity of 595 core GR binding sites aligned according to their summits. Three clusters were applied, and 5 kb upstream and 5 kb downstream regions around the summit are plotted. (E) UCSC browser tracks for TSC22D3, FKBP5 and MT1X in normoxia and hypoxia, where the y axis of each track represents the coverage by non redundant and extended reads from MACS analysis. These RefSeq annotated genes are shown and the gene direction indicated by an arrowhead. GR binding sites were quantified by ChIP-qPCR. Graphs depict fold enrichment of immunoprecipitated GR in response to treatment with dexamethasone over vehicle treated control and show mean ± S.D. from three independent experiments. *p < 0.01, NS: not significant.