Figure 3: Wap super-enhancer activates the Ramp3 gene.

(a) RNA-seq data from mammary tissue at day 6 of pregnancy (p6) and day 1 of lactation (L1) demonstrated that Wap and Ramp3 are induced during pregnancy, although Ramp3 is expressed at a much lower level. Tbrg4 expression remained unchanged (GSE37646). Results are shown as box plots; median, middle bar inside the box; IQR, 50% of the data; whiskers, 1.5 times the IQR; p6: n=3 and L1: n=2. Student’s t-test with alpha 0.01 was applied for p6 and L1 for each gene independently. *P<0.01, **P<0.001, ***P<0.0001. (b) The cartoon illustrates Wap and its flanking genes. The blue circles represent the constituent enhancers of the super-enhancer (shown as red bar). H3K27ac is shown in green. The crimson ovals represent the sites bound by CTCF. (c) Gene expression data from the enhancer mutant ΔS3 demonstrated that Ramp3 expression was reduced by 70%. Results are shown as the means±s.e.m. of independent biological replicates; ΔS3: n=3; data were normalized to Gapdh; a two-tailed Student’s t-test was applied with alpha 0.01 for wild type and ΔS3 for each gene independently. *P<0.01, **P<0.001, ***P<0.0001. (d) STAT5 binding and H3K27ac in the Wap-Ramp3 locus in WT and ΔS3 mutant mammary tissue (GSE74826). (e) H3K27ac at the Wap-Ramp3 locus in mammary tissue, Th17 cells and adipocytes (GSE90788, GSE92590). (f) H3K27ac at the Il2rα locus in mammary tissue, Th17 cells and adipocytes. (g) H3K27ac at the Mir193b locus in mammary tissue, Th17 cells and adipocytes.