Fig. 2

Identification and analysis of the SOX9 enhancer eSR-B. a Schematic of a number of previously published 46,XX DSD duplication/triplication (grey)^16,18,20,21 and 46,XY DSD deletion (blue)^17,22 patients upstream of SOX9. The minimal overlap (RevSex) of the six patients (24 kb) in magenta. b Unbiased luciferase tiling strategy of the minimal RevSex region (16 constructs—blue) to test for enhancer activity using the PGL4-βg vector. c Two fragments, b8 and b9, showed significant enhancer activity and their overlapping fragment, eSR-B was sub-cloned (blue lines). A conserved SOX9 binding site was identified (magenta line and box) and UCSC genome browser ENCODE data (HMEC) highlighted a strong active enhancer site (orange), conservation tract indicated a conserved sequence (blue peaks). No DNaseI peaks were detected in human foetal testis or ovaries (green). d Enhancer activities of small fragments (b1-b16) of the minimal overlap in COS7 cells transfected with SF1 and SOX9 in luciferase assays. Only the fragments b8 and b9 showed significant enhancer activity compared to the empty vector PGL4-βg (n = 3). e Enhancer activities of b8 and b9 overlapping region eSR-B as assessed by luciferase assays in COS7 cells co-transfected with SF1 (white bars) and SOX9 alone (grey bars) and in combination (blue bars) (n = 4). Significance is compared to the PGL4-βg empty vector control. f Enhancer activity of eSR-B fragment with mutation of the SOX9 (eSR-B ΔSOX9) binding site motif in response to SOX9 transfection (n = 7) significance compared to the eSR-B non-mutated fragment. g Luciferase reporter assays show FOXL2 transfected with SOX9 represses eSR-B enhancer activity compared to SOX9 alone (n = 4). All Luciferase assays are carried out in COS7 cells. Error bars are s.e.m. P-values derived from two-tailed t tests: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a Source Data file.