Figure 7. HIF1α is required for naïve to primed hESC transition.
A: screen shot of RNA expression and H3K27me3 marks of EGLN1 (PHD2) in naïve hESCs [Elf112, WIRB3 naïve and BGO1 naïve8)], primed hESCs [WIRB3 primed8, H1 and H964 and Elf1 treated with STAT3 inhbitor (100 µM) for 6h. B: HIFα is hydroxylated on prolyl residues by EGLN1 (PHD2), leading to VHL-mediated proteolysis. C-D: Sequencing trace files, DNA sequences and protein sequences of HIF1α CRISPR-Cas9 knock-out (KO) mutant clones (gHIF1 6.2.1, C; gHIF1 6.3.1, D). E: schematic representation of wild type HIF1α protein and proteins resulting from CRISPR-Cas9 knock-out (KO) mutants gHIF1 6.2.1 and gHIF1 6.3.1. bHLH= basic helix-loop-helix domain, PAS= Per-Arnt-Sim domain, NTAD= N-terminus transcriptional activation domain, CTAD= C-terminus transcriptional activation domain. F: HIF1α is not expressed in CRISPR-Cas9 KO mutants. Western blot analysis of HIF1α expression in cells pushed toward the primed stage by culture in TeSR1 for 5 days in wild type Elf1 cells (iCas9 Elf1), and two CRISPR-Cas9 KO mutants of HIF1α (gHIF1 6.2.1 and gHIF1 6.3.1). G: qPCR analysis of hESCs transitioning to primed reveals that naïve markers (DNMT3L and NNMT) are still expressed higher in Elf1 HIF1α CRISPR-Cas9 KO cells compared to wild type Elf1, while primed marker IDO1 and HIF target genes (PDK1 and VEGFA) are downregulated (n=3; s.e.m.; p=0.024 for DNMT3L, p=0.0005 for NNMT, p=0.001 for IDO1, p=0.12 for PDK1, p=0.004 for VEGFA; 2-tailed t-test). H: KO of HIF1α prevents the metabolic switch occurring during the transition of hESCs from naïve to primed state as shown by measuring OCR after FCCP addition using SeaHorse. n=3 for gHIF1 6.3.1 2iLIF and AF and n=4 for Elf iCas9 and gHIF1 6.2.1 2iLIF and AF; s.e.m.; p=0.0117 for gHIF1 6.2.1 vs. Elf iCas9, p=0.0032 for gHIF1 6.3.1 vs. Elf iCas9; 2-tailed t-test. Unprocessed original scans of blots are shown in Supplementary Fig.9. For raw data, see Supplementary Table 4. n= number of biological replicates.