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Comparative Study
. 2006 Mar 1;20(5):557-70.
doi: 10.1101/gad.1399906.

HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

Kelly L Covello  1 , James Kehler, Hongwei Yu, John D Gordan, Andrew M Arsham, Cheng-Jun Hu, Patricia A Labosky, M Celeste Simon, Brian Keith
Affiliations
Comparative Study

HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

Kelly L Covello et al. Genes Dev. .

Abstract

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1alpha with HIF-2alpha results in expanded expression of HIF-2alpha-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2alpha, but not HIF-1alpha, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2alpha KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2alpha severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2alpha-specific target gene and indicate that HIF-2alpha can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2alpha's tumor promoting activity.

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Figures

Figure 1.
Figure 1.
Disrupted development in Hif-2α KI embryos. (A–F) Between E10.5 and E13.5, ∼50% of Hif-2α KI/+ heterozygous embryos demonstrated a range of phenotypes. At E13.5, Hif-2α KI/+ heterozygous yolk sacs and embryos display hemorrhaging (B, arrows) and gross abnormalities (C), compared with wild-type (WT) controls (A). (C) Several embryos disintegrated upon dissection, suggesting they were dead or severely abnormal. At E10.5, Hif-2α KI/+ heterozygous embryos displayed phenotypes ranging from subtle hemorrhaging (E, arrow) to cardiac effusion (F, asterisk). A wild-type (WT) E10.5 embryo control is shown in D. (G–K) Comparison of wild-type (WT) (G,H) and homozygous Hif-2α KI/KI (I) and presumed homozygous Hif-2α KI/? (J,K) E7.5 embryos. Genotypes of embryos in J and K were inferred from morphological similarity to confirmed Hif-2α KI/KI embryos (shown in I) and frequency (10% of total embryos). Hif-2α KI/KI embryos displayed improper formation of the epiblast (ep, arrows in H,J,K), and embryonic (em) and extraembryonic (ex) cavities, compared with wild type (WT). (L–P) Whole-mount RNA in situ hybridization on E7.5 embryos using probes for the mesodermal marker Brachyury (Bry) (L,M) and visceral endodermal marker α-fetoprotein (AFP) (N–P). In contrast to wild-type (WT) littermate controls (L,N), which display normal mesoderm migration through the primitive streak (bracket in L), Hif-2α KI/KI embryos exhibit patterning defects (M) consistent with abnormal gastrulation. Similarly, AFP expression is restricted to the extraembryonic visceral endoderm in wild-type embryos (arrow in N), but maintained in embryonic visceral endoderm in Hif-2α KI/KI embryos (O,P).
Figure 2.
Figure 2.
Increased Oct-4, Vegf, and Tgf-α expression in Hif-2α KI/KI embryos. (A) Genotypes of individual E7.5 embryos were determined by PCR on genomic DNA and quantitative RT–PCR analysis of Hif-1α, Hif-2α, and Pgk transcripts. Transcript levels were normalized to 18S rRNA transcripts in each sample. Data are expressed as the ratio of target gene expression in each sample relative to a known wild-type (WT) litter (n = 8) that was stage matched. (B) Average levels of Hif-1α and Hif-2α mRNA in embryos pooled by genotype. (C) Average levels of Oct-4, Vegf, and Tgf-α mRNA in embryos pooled by genotype. (D–F) Whole-mount RNA in situ hybridization for Oct-4 mRNA demonstrated that Oct-4 is expressed in the embryonic epiblast in both wild-type (WT) and homozygous Hif-2α KI/KI E7.5 embryos.
Figure 3.
Figure 3.
Increased Vegf and Tgf-α expression in Hif-2α KI/KI 3.5 d EBs. (A) Schematic of HIF-1α control knock-in allele, in which a c-Myc-tagged Hif-1α cDNA was targeted to the first coding exon of the murine Hif-1α locus in ES cells. A detailed description is presented in Supplementary Figure 2. (B) Extracts from homozygous Hif-1α KI/KI and Hif-2α KI/KI ES cells were immunoprecipitated with c-Myc epitope antibodies and then analyzed on Western blots using c-Myc antibodies. Western blot analysis using AKT antibodies confirms that each sample in the immunoprecipitation experiment had equal amounts of input protein. (N) Normoxia (21% O2); (H) hypoxia (4 h at 1.5% O2). (C) Quantitative RT–PCR analysis in 3.5-d EBs of Alda A transcripts in wild-type (WT) and homozygous Hif-1α KI/KI, Hif-2α KI/KI, and Hif-1α−/− EBs. Fold change indicates increases in mRNA in EBs grown at 3.0% O2 relative to EBs grown at 21% O2. Transcript levels were normalized to 18S rRNA transcripts in each sample. (D) Real-time PCR analysis in 3.5-d EBs cultured at 21% O2 indicates increased Tgf-α, Vegf, and Hif-2α mRNA in two independently derived homozygous Hif-2α KI/KI clones. Changes in expression are specific to the Hif-2α KI allele, as Hif-1α KI/KI clones and Hif-1α−/− EBs behave similarly to wild type. (*) p < 0.005.
Figure 4.
Figure 4.
Expanded HIF-2α expression promotes Oct-4 expression and activity in 3.5-d EBs, and HIF-2α protein directly binds the Oct-4 promoter. (A) Real-time PCR analysis of Oct-4 demonstrates increased Oct-4 mRNA in normoxic 3.5-d EBs generated from two separately derived homozygous Hif-2α KI/KI clones. Increased Oct-4 expression is specific to the Hif-2α KI/KI EBs, as Hif-1α KI/KI and Hif-1α−/− EBs behave similarly to wild type (WT). (*) p < 0.005. (B) Western blot analysis shows increased Oct-4 protein in independent homozygous Hif-2α KI/KI EB clones. Levels were normalized to the AKT loading control, and fold change was determined by densitometry. (C) Real-time PCR analysis for mesodermal markers Fgf-4 and Bry mRNA demonstrates elevation of Oct-4 target genes specifically in independent homozygous Hif-2α KI/KI EBs. (*) p < 0.005. (D) Schematic diagram of the Oct-4 promoter displaying the relative position of putative HREs, as well as regions of notable sequence conservation between mouse and human (CR1–CR4). (E) ChIP on 293 cells shows that HIF-2α binds the putative HREs in CR4 and CR3 of the Oct-4 promoter. PCR products are obtained only when immunoprecipitated with HIF-2α and not when immunoprecipitated with an isotype control nonspecific antibody. No PCR product was observed in the HIF-2α ChIP using prim ers between CR2 and CR1 of the Oct-4 gene. Furthermore, HIF-1α does not appear to bind these regions in the Oct-4 promoter. (F) Transient transfection of Oct-4 promoter reporters into 786-O WT-8 renal carcinoma cells demonstrates that Oct-4 is induced by hypoxia (1.5% O2), but this induction is abolished when the HREs in CR4 and CR3 are removed. Normoxic basal expression was unchanged when comparing full-length Oct-4 promoter constructs (GOF-9) and Oct-4 promoter constructs with the HREs deleted (GOF-6).
Figure 5.
Figure 5.
Reversal of Hif-2α KI hematopoietic phenotypes by TGF-α and Oct-4 inhibition. (A) Normoxic day 9 Hif-2α KI/KI EBs form fewer hematopoietic CFUs than wild-type (WT) EBs. Numbers of CFU-E, CFU-M, CFU-G, and CFU-GM, CFU-GEMM progenitors are shown. Homozygous Hif-2α KI/KI EBs form more secondary EBs, compared with wild type. (B) Comparison of total CFU formation in EBs derived from ES cells of different genotype, expressed as a percentage of wild type (WT). Note the statistically significant decrease in the ability of homozygous Hif-2α KI/KI ES cells to form CFUs. This phenotype is specific to the two independent homozygous Hif-2α KI/KI clones, as Hif-1α KI/KI clones, Hif-1α−/− EBs, and Hif-2α KI/KI + HIF-2α shRNA clones behave similarly to wild type. (*) p < 0.005. (C) Western blot analysis for phosphorylated EGFR revealed inhibition of TGF-α activity by PD153035 in wild-type (WT) and in homozygous Hif-2α KI EBs. Hif-2α KI/KI EBs exhibited hyperphosphorylation of EGFR, consistent with increased TGF-α activity. Reduction in EGFR phosphorylation in wild-type EBs correlated to a decrease in total CFU formation. In contrast, reduction of EGFR phosphorylation in Hif-2α KI/KI EBs correlated to increased CFU formation. Restoration of CFU formation in Hif-2α KI/KI EBs was most notable in samples displaying nearly wild-type levels of EGFR phosphorylation. Total CFU formation is expressed as a percentage of that obtained with EBs derived from wild-type ES cells. (*) p < 0.005. (D) Western blot analysis indicating variable knock-down of Oct-4 protein and activity (Fgf-4 RNA expression) in EBs derived from homozygous Hif-2α KI/KI ES cell clones expressing Oct-4 shRNAs. EBs derived from Hif-2α KI/KI ES clones with Oct-4 protein levels similar to wild type (WT) displayed reduced expression of Fgf-4 RNA, and partial restoration of CFU formation compared with Hif-2α KI/KI. (*) p < 0.005. (E) EBs derived from Hif-2α KI/KI ES clones doubly inhibited for Oct-4 and TGF-α show even greater restoration of CFU formation and reduced secondary EB formation compared with Hif-2α KI/KI.
Figure 6.
Figure 6.
HIF-2α mediates tumor growth through up-regulation of Oct-4. (A) Gross appearance of teratomas resected from nude mice 21 d after subcutaneous injection with willd-type (WT), Hif-2α KI/KI ES cells, and Hif-2α KI/KI ES cells with Oct-4 shRNA knock-down (Hif-2α KI/KI-Oct-4 shRNA). (B) Final teratoma masses of wild-type (WT), Hif-2α KI/KI, and Hif-2α KI/KI-Oct-4 shRNA teratomas formed after 22 d of growth. Error bars measure standard error of the mean. (*) p = 0.012. (C) Western blot analysis of Oct-4 and β-catenin protein in wild-type (WT), Hif-2α KI/KI, and Hif-2α KI/KI-Oct-4 shRNA teratomas. Data are representative of three independent teratomas per genotype. (D) Ki67 staining of wild-type (WT), Hif-2α KI/KI, and Hif-2α KI/KI-Oct-4 shRNA teratoma sections. Final magnifications are 200×. (E) Nanog staining of wild type (WT), Hif-2α KI/KI, and Hif-2α KI/KI with Oct-4 shRNA knock-down teratoma sections.
Figure 7.
Figure 7.
HIF-2α is required for Oct-4 expression and normal PGC numbers. (A) Real-time PCR analysis demonstrates increased hypoxic induction (1.5% O2 for 16 h) of Oct-4 mRNA in wild-type (WT) and Hif-1α−/− 3.5-d EBs, which is diminished in Hif-2α−/− EBs. (B–D) PGCs in E8.5 embryos revealed by AP staining. Arrows point to representative PGCs in C and D. In contrast to wild-type (WT) and heterozygous Hif-2α+/− littermate controls (C), somite-matched Hif-2α−/− embryos exhibit reduced numbers of PGCs (D). (B) Quantification of PGC numbers in somite-matched E8.5 (s7–s11) embryos. (E,F) PGCs in E12.5 genital ridges dissected from embryos revealed by AP staining. (G) Proposed model for regulation of Oct-4 by HIF-2α. In PGCs, the Oct-4 locus is expressed and regulated by HIF-2α. The Oct-4 locus is also expressed in ES cells; however, HIF-2α activity is normally restricted or repressed in these cells (C.-J. Hu, S. Iyer, A. Sataur, K.L. Covello and M.C. Simon, in prep.). In differentiating somatic cells, the Oct-4 locus adopts a closed conformation and is not expressed. At this point, HIF-2α regulates other target genes (e.g., Vegf) without modulating Oct-4 levels. In homozygous Hif-2α KI/KI embryos, EBs, and teratomas, expanded HIF-2α expression results in up-regulation of Oct-4 and other targets. HIF-2α-mediated expression of an inappropriately derepressed Oct-4 locus in transformed cells could similarly modulate cancer stem cell identity and tumor progression.
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