Figure 8: Border CTCF site is required for Ramp3 expression in non-mammary cells.

(a) H3K4me3 marks at the Ramp3 promoter indicated active transcription in several non-mammary cell types and tissues (GSE29218, GSE31039, GSE32864). (b) Out of the three CTCF-binding sites in mammary tissue that separate the Wap super-enhancer and Ramp3, only site E (filled crimson ovals) was present in non-mammary tissue, like cerebellum, heart, kidney and uterus. CTCF binding to sites C and D was absent in those tissues (dashed crimson ovals) (c) Deletion of site E as well as the combined deletion of sites C, D and E led to a 90% reduction in Ramp3 expression in the cerebellum. Deletion of site D did not affect Ramp3 expression. Results are shown as the means±s.e.m. of independent biological replicates; ΔD: n=3 (−/−); ΔE: n=6 (−/−); ΔCDE: n=4 (−/−); data were normalized to Gapdh; one-way ANOVA with multiple comparison and alpha 0.01 was applied. *P<0.01, **P<0.001, ***P<0.0001. (d) Deletion of site E led to a significant reduction in Ramp3 expression in several non-mammary cells. Results are shown as the means±s.e.m. of independent biological replicates; Kidney and Uterus ΔE: n=3 (−/−); Heart ΔE: n=4 (−/−); data were normalized to Gapdh; a two-tailed Student’s t-test with alpha 0.01 was applied. *P<0.01, **P<0.001, ***P<0.0001. (e) CTCF site E coincided with DNase I hypersensitivity. Sites C and D were not occupied by CTCF in non-mammary cells and no DNase I hypersensitivity had been detected at these sites (GSE37074).