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. Author manuscript; available in PMC: 2023 May 25.
Published in final edited form as: Circulation. 2023 Apr 17;147(21):1606–1621. doi: 10.1161/CIRCULATIONAHA.122.061940

Figure 1). Defining Upstream Regulators of SOX17 & Effects of Common Variation at the SOX17 Locus in PAH.

Figure 1)

A) Knockdown of SOX17 through CRISPR-Inhibition of SOX17-signal 2. Relative gene expression of SOX17 compared to ACTβ in hPAEC. Ordinary 1-way ANOVA of conditions compared to BFP condition with Dunnett’s multiple comparisons test. n=3 experiments performed in triplicate. B) Knockdown of SOX17 through CRISPR-deletion of SOX17-signal 1 region. Relative gene expression of SOX17 compared to ACTβ in hPAEC. CRISPR-deletion guides A/B target SOX17-signal 1. Ordinary 1-way ANOVA of conditions compared to BFP condition with Dunnett’s multiple comparisons test. n=3. C) EMSA assay showing binding of hPAEC nuclear proteins to 21bp DNA probes containing the sequence at the rs1098403 region. Shift and supershift are highlighted. H indicates cold probes containing a putative HOXa5 binding site. Biotin-probe, biotin-labelled probe (G/A, alleles of SOX17 enhancer variant included in probe sequence). Cold-Probe, unlabelled probe. H, HOXa5 competitive probe. IgG, Mouse IgG. The black triangles show increasing molecular excess from left to right. D) RNAseq expression in hPAECs (RPKM) of potential TFs of interest for rs1098403. HOXa5 is shown in red. SOX17 is shown in black for reference. All other transcription factors are shown in blue and were found through CIS-BP. The binding score (taken from CIS-BP) refers to the predicted likelihood of the transcription factor binding to the given sequence and decreases from left to right. The underlined sequence refers to the potential binding location of HOXa5 and the A in red font is the site of rs1098403. The consensus binding sequence of HOXA5 was taken from JASPAR (jaspar.genereg.net) and is shown with the sequence of the region surrounding rs1098403 and rs765727 were taken from https://genome.ucsc.edu/. *-p<0.05, **-p<0.01. n=3. E) ChIP-qPCR for HOXA5 at rs10958493. Results of a quantitative PCR (triplet measurements per donor, n=3) performed on precipitated fraction of chromatin immunoprecipitation (ChIP) with IgG or HOXa5 antibody in 4 HPAEC donors. The ChIP with IgG control was performed in HPAEC donor C19.