Fig. 6.

Functional testing of putative enhancers by transient transfection. a Tracks for RNA-seq, POU1F1, H3K27Ac, H3K4Me1, ATAC-seq, and H3K27Me3 are shown for Gata2 in TαT1 (red) and GHF-T1 (blue) cells. The putative enhancer regions cloned for the luciferase assay are highlighted in red, orange, green, light blue, and dark blue. b The level of luciferase activity of each element relative to the smallest promoter region is shown. Prom 1, 2, and 3 represent the Gata2 promoter with 0.2, 0.9, and 2.8 kb of 5′ flanking region, respectively. 1, 2, and 3 represent the three similarly highlighted elements in a tested in both the forward (circles) and reverse (x’s) orientation upstream of the 0.2 kb Gata2 promoter. The significance was evaluated with a two-sided t-test and indicated with asterisks where p value < 0.05 = * and p value < 0.01 = **. c Same tracks as in a, at the Cga locus, where elements tested are highlighted. d Level of luciferase activity of each element, color-coordinated with the highlighted elements in c in both the forward (circles) and reverse (x’s) orientation. e Same tracks as in a at the Pitx1 locus, where elements tested are highlighted. f Level of luciferase activity of each element, color-coordinated with the highlighted elements in f. Elements were tested only in the forward orientation. N = 6 replicates/reporter gene