Figure 2
TFAP2A can regulate HIPSTR promoter, but HIPSTR and TFAP2A are not consistently co-induced in in vitro developmental models.
(A) Re-analyses of H3K4me3 ChIP-seq data from refs 42, 43, 44, 45 reveal conserved HIPSTR promoter demarcation across the genomes of 10 mammalian species (see Methods) and of chicken, and absence of H3K4me3 mark around HIPSTR TSS orthologous region in frog and zebrafish. The maximal value on the y-axis scale corresponds to the highest H3K4me3 peak across the entire TFAP2A locus for each species. (B) Positions of the mapped H3K4me3 and TFAP2A ChIP-seq reads from ref. 29 (HeLa-S3 cells) and ref. 24 (three human NCC and two chimp NCC lines), and positions of the DNA sequences used for HIPSTR promoter-reporter assays (pGL3-P1 to -P7) relative to the TFAP2A locus genes. (C) Luciferase reporter assays in HEK293 cells upon TFAP2A isoform 1a overexpression. DNA sequences surrounding HIPSTR TSS (see above) were cloned upstream of the firefly luciferase gene, and co-transfected with plasmid overexpressing TFAP2A isoform 1a or with empty vector; pGL3-Basic served as negative control (no promoter upstream of the firefly luciferase); pGL3-SV40 served as positive control (SV40 promoter upstream of the firefly luciferase); 3xAP2bluc served as positive control for transactivation by TFAP2A isoform 1a. (D) Overexpression of TFAP2A isoforms 1a (dark red), 1b (red), or 1c (pink) upregulates endogenous HIPSTR in HEK293 cells, as measured with RT-qPCR. (E–G) HIPSTR is moderately co-upregulated with TFAP2A in in vitro derived human NCCs (E), weakly co-upregulated with TFAP2A in in vitro derived human TBCs (F), and not co-upregulated with TFAP2A in NT2/D1 cells treated with ATRA, where HIPSTR remains undetectable (G), as measured with RT-qPCR. Upregulation of TFAP2A gene itself (NCCs marker), of CGB (human TBCs marker), or HOXB5 gene (induced by ATRA treatment in NT2/D1 cells)47 served as positive controls in the corresponding experiments shown on (E–G). Experiments presented on (C–G) were performed in triplicate, and error bars represent SD. For experiments on (C,D) the asterisks indicate statistical significance of the observed changes calculated with two-tailed t-test, equal variance (p-value < 0.01).
