Figure 5.

Capacity of Wap super-enhancer elements to activate the common Ramp3 gene. (A) Diagram of mutants in which Wap enhancers were fused with the Ramp3 promoter (E12-R3 and E2-R3). In E2-R3 mutants, the GAS site in E1 was deleted and was marked in gray. By deleting a 17.8 kb stretch of DNA, the Wap promoter and enhancers E1 and E2 were translocated within 600 bp of the Ramp3 transcription start site. The exact positions of the deletions are shown in Supplementary Table S9. (B) STAT5A, RNA Pol II and H3K27ac landscapes at the Wap-Ramp3 locus in WT, ΔE3 and E12-R3 mutant tissue at day 10 of lactation (L10) as well as the CTCF and H3K4me3 landscapes in WT mammary tissue at day 1 of lactation (L1). H3K27ac expansion and RNA Pol II loading occurred across the breakpoint into the Ramp3 promoter and gene body (red asterisks). The Lalba locus was used as a control for ChIP-seq quality (Supplementary Figure S7). (C) RNA Pol II and H3K27ac coverage of the Ramp3 promoter region in WT and mutant mammary tissue. (DandE) Ramp3 (D) and Wap (E) mRNA levels in mammary tissues from ΔE3 and E12-R3 mutants at L1 and L10 were measured by qRT-PCR and normalized to Gapdh levels. Results are shown as the means ± SEM of independent biological replicates (ΔE3 at L1 and L10, E12-R3 at L1 and L10, n = 3). ANOVA was used to evaluate the statistical significance of differences between ΔE3 and E12-R3 mutant mice. **P < 0.001, ***P< 0.0001, ****P < 0.00001. n.s. not significant.