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. 2021 Dec 8;6(23):e150059. doi: 10.1172/jci.insight.150059

Figure 5. GATA4 is a direct repressor of EPAS1 in HSCs.

Figure 5

(A) Quantitative RT-PCR analysis of EPAS1 expression in LX2 cells transfected with GFP-expressing (Ad-GFP) and GATA4-expressing (Ad-GATA4) adenoviruses (n = 3, Ad-GFP; n = 4, Ad-GATA4). (B) Quantitative RT-PCR analysis of EPAS1 expression in G2-Cre;Gata4 KO E13.5 embryonic liver (n = 5, control; n = 4, GATA4 KO). (C) Quantitative RT-PCR analysis of EPAS1 expression in livers of adult Gata4-floxed mice injected with GFP-expressing (Ad-GFP) (n = 4) and Cre-expressing adenoviruses (Ad-Cre) (n = 3). (D) Quantitative RT-PCR analysis in LX2 cells transfected with a siRNA directed against EPAS1 compared with control LX2 cells (treated with siRNA-negative control) (n = 3). (E) Schematic of the human EPAS1 intronic region containing 2 conserved GATA sites using Vista Tools software. The blue peak indicates the human EPAS1 transcription start, and the pink peaks indicate conserved noncoding regions between human and mouse. An 810 bp fragment of EPAS1 intronic region containing the 2 conserved GATA4 sites (EPAS1 wt) or the mutated version (EPAS1 mut) was cloned into the pGL3 luciferase vector for reporter assays. The nucleotides mutated in EPAS1 mut are shown in lowercase. The numbers indicate the localization in the EPAS1 locus from the transcriptional start site. (F) ChIP of LX2-overexpressing GATA4 using a GATA4-specific antibody or a IgG-unspecific antibody (n = 3). (G) In vitro luciferase reporter assays in 293T cells of pGL3-EPAS1 wt and pGL3-EPAS1 mut plasmids (n = 3). Statistical analyses was performed using 2-tailed Student’s test. Error bars represent mean ± SEM. *P < 0.05. RLU, relative light units.