Figure 1.
HIF-2α directly binds to HREs in the OCT4, NANOG and SOX2 proximal promoters in hESCs cultured under hypoxia.
(A) ChIP analysis of HIF-2α binding a predicted HRE site in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G on chromatin isolated from hESCs cultured either at 20% or 5% O2. Amplification of FOXP3 was used as a negative control. DNA enrichment is expressed as a percentage of Input. An average of 3 independent experiments is represented (*P<0.05, **P<0.01; NS: no significant difference). (B) Schematic representation of the HIF-2α expression vector (pcDNA3.1-HIF-2α top panel). The luciferase reporter construct driven by NANOG promoter showed a significant increase (**P<0.01) in luciferase activity compared to the control. Mutation in the predicted HRE caused a significant decrease in the luciferase activity (**P<0.01) compared to the control. An average of 4 independent experiments is shown.
Figure 2.
Hypoxia and reoxygenation enhance the expression of pluripotency genes through a euchromatic state within the HRE site.
(A) RT-qPCR analysis for OCT4, NANOG and SOX2 mRNA in hESCs subjected to 24, 48 and 72 hours reoxygenation compared to those maintained at 20% O2. All data have been normalized to UBC and to 1 for hESCs maintained at 20% O2. Values are mean of 5 independent experiments ± SEM (*P<0.05, **P<0.01, ***P<0.001 significantly different from 20% O2). (B) Pie charts showing ChIP analysis of histone modification markers H3K4me3, H3K36me3 and H3K9me3 binding a predicted HRE site in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G in hESCs exposed to 20% O2, 5% O2, and hypoxia reoxygenation respectively. DNA enrichment is expressed as a percentage of Input relative to the IgG control. An average of 3 to 4 independent experiments is represented.
Figure 3.
HIF-2α sustains the expression of NANOG following hypoxia/reoxygenation.
ChIP analysis of HIF-2α binding a predicted HRE site in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G on chromatin isolated from hESCs subjected to hypoxia followed by 72 hours reoxygenation. HIF-2α binding to the HRE of pluripotency genes is expressed relative to 20% O2 (A) or 5% O2 (B). DNA enrichment is expressed as a percentage of Input. An average of 4 independent experiments is represented (*P<0.05, ***P<0.001). RT-qPCR for HIF-2α expression in hESCs subjected to 24, 48 and 72 hours reoxygenation. All data have been normalized to UBC and to 1 for hESCs cultured at 20% O2 (C) or 5% O2 (D). Values are mean of 3 to 4 independent experiments ± SEM (*P<0.05).
Figure 4.
Hypoxia/reoxygenation induces HIF-2α binding to the oct-sox cis element in the NANOG promoter.
(A) Schematic representation of the probes designed to cover the HRE, oct-sox cis element and intermediate region on the NANOG proximal promoter. (B) ChIP analysis of HIF-2α binding to the oct-sox-cis element and the intermediate region in hESCs cultured under hypoxia followed by 72 hours reoxygenation. An average of 4 independent experiments is represented (*P<0.05, **P<0.01). ChIP analysis of HIF-2α binding the oct-sox cis-regulatory element in NANOG proximal promoter on chromatin isolated from hESCs cultured at 5% O2 (C), 20% O2 (D). ChIP analysis of HIF-2α binding the oct-sox cis-regulatory element in OCT4 proximal promoter (E) and SOX2 intron (F) on chromatin isolated from hESCs subjected to 72 hours reoxygenation. DNA enrichment is expressed as a percentage of Input. An average of 3 independent experiments is represented. (NS: no significant difference).
Figure 5.
HIF-2α binds the oct-sox cis-regulatory element and drives NANOG activity.
Schematic representation of the HIF-2α expression vector (pcDNA3.1-HIF-2α top panel). A pcDNA3.1-HIF-2α vector was transiently co-transfected in NT2 cells with a luciferase reporter construct driven by NANOG promoter with intact HRE and oct-sox element or with different constructs in which either the HRE, the oct-sox or both sites were mutated. Luciferase activity was significantly decreased when the HRE (P<0.01), the oct-sox element (P<0.01) and both sites (P<0.001) were mutated compared to the NANOG gene promoter construct with the unmutated HRE and oct-sox element. An average of 4 independent experiments is shown.
Figure 6.
OCT4 and SOX2 bind to the oct-sox cis element in the NANOG proximal promoter.
ChIP analysis of OCT4 and SOX2 binding to the oct-sox cis element in hESCs cultured at either 5% O2, 20% O2, or 5% O2 followed by 72 hours reoxygenation. An average of 4 independent experiments is represented (**P<0.01, ***P<0.001).
Figure 7.
Schematic representation of NANOG promoter regulation under conditions of normoxia, hypoxia and reoxygenation in hESCs.