HIF-2α binds the oct-sox <i>cis</i>-regulatory element and drives NANOG activity.

<p>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.</p

Hypoxia and reoxygenation enhance the expression of pluripotency genes through a euchromatic state within the HRE site.

<p>(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.</p

Hypoxia/reoxygenation induces HIF-2α binding to the oct-sox <i>cis</i> element in the NANOG promoter.

<p>(A) Schematic representation of the probes designed to cover the HRE, oct-sox <i>cis</i> element and intermediate region on the NANOG proximal promoter. (B) ChIP analysis of HIF-2α binding to the oct-sox-<i>cis</i> 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 <i>cis</i>-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 <i>cis</i>-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).</p

Schematic representation of NANOG promoter regulation under conditions of normoxia, hypoxia and reoxygenation in hESCs.

<p>Schematic representation of NANOG promoter regulation under conditions of normoxia, hypoxia and reoxygenation in hESCs.</p

HIF-2α sustains the expression of NANOG following hypoxia/reoxygenation.

<p>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).</p

HIF-2α directly binds to HREs in the OCT4, NANOG and SOX2 proximal promoters in hESCs cultured under hypoxia.

<p>(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.</p

Hypoxic culture promotes glucose uptake and lactate production in hESCs.

<p>Glucose, pyruvate and lactate utilisation were non-invasively measured in a defined hESC medium. More glucose was consumed and lactate produced by Hues7 hESCs cultured at 5% O₂ than at 20% O₂ on (A) day 2 (B) day 3 and (C) day 4 post-passage. In contrast, less pyruvate was consumed by hESCs at 5% O₂ compared to 20% O₂. The rate of glucose consumption and lactate production was also greater on day 3 post-passage in Shef3 hESCs cultured at 5% O₂ compared to those maintained at 20% O₂ (D). **P<0.01, ***P<0.001 significantly different to 5% O₂ (n = 12–23).</p

GLUT1 expression parallels glucose utilisation and is directly regulated by HIF-2α under hypoxic conditions.

<p>RT-qPCR was used to quantify <i>GLUT1</i> mRNA expression in Hues7 hESCs cultured at either 5% O₂, or 20% O₂ on day three post-passage (A). All data has been normalised to <i>UBC</i> and to 1 for 5% O₂. *P<0.05 significantly different to 5% O₂ (n = 3). Using siRNA to silence HIF-α subunits in Hues7 hESCs cultured at 5% O₂, <i>GLUT1</i> mRNA was found to be regulated by HIF-2α (B). All data has been normalised to <i>UBC</i> and to 1 for the transfection control. *P<0.05 significantly different to transfection control (n = 6). Using ChIP HIF-2α was found to bind to the proximal promoter of GLUT1 only in hESCs cultured at 5% O₂. ChIP assays were performed with either a HIF-2α or IgG control antibody on chromatin isolated from Hues7 hESCs cultured at either 20% O₂ or 5% O₂. DNA enrichment is expressed as a percentage of input (non-immunoprecipitated chromatin). *P<0.05, **P<0.01, NS indicates no significant difference (n = 5).</p

hESCs maintained at atmospheric O₂ levels express reduced levels of pluripotency markers compared to those cultured at 5% O₂.

<p>Hues7 hESCs were cultured at either 5% O₂, 5% O₂ with FGF2 removed for 16 hours (5% O₂– FGF2) or 20% O₂. Protein was isolated and OCT4 (A and B), SOX2 (C and D) and NANOG (E and F) quantified using Western blotting. All data has been normalised to β-actin and to 1 for 5% O₂. *P<0.05, **P<0.01, ***P<0.001 significantly different from 5% O₂ (n = 3–4).</p