Fig. 3

Identification and analysis of the SOX9 enhancer eALDI. a Schematic of the upstream region of SOX9 highlighting human TESCO (hTESCO) and the proposed enhancer eALDI. Bioinformatic data from UCSC genome browser. ENCODE data (HMEC) indicated a strong active enhancer (orange) covering the proposed eALDI region. DNaseI peaks were observed in one foetal testis track (dark green), and strong conservation across the region was observed (blue peaks). A predicted SRY transcription factor binding motif is presented by magenta vertical line and box. By comparison ENCODE and DNaseI data for hTESCO show less enhancer potential and lower levels of conservation. b Enhancer activity of eALDI and hTESCO measured by luciferase assays in COS7 cells when co-transfected with SOX9 alone (grey bars), SOX9 + SF1 (blue bars) and SF1 + SRY (green bars). The eALDI region shows significant enhancer activity in response to both SF1 + SOX9 and SF1 + SRY transfection when compared to the empty pgl4-βg (n = 4). c Mutation of the SRY (eALDIΔSRY) transcription factor motif in the eALDI enhancer reduced activity when co-transfected with SF1 + SOX9 or SF1 + SRY compared to unmuted eALDI. Error bars represent s.e.m. P-values derived from two-tailed t tests: **P ≤ 0.01, ****P ≤ 0.0001. d Luciferase reporter assays in COS7 show FOXL2 transfected with SF1 + SOX9 represses eALDI enhancer activity compared to co-transfection with SOX9 + SF1 alone (n = 3) Error bars are s.e.m. P-values derived from two-tailed t tests: ****P ≤ 0.0001. e CRISPR/Cas9 mediated knock-out of eALDI resulted in reduction of Sox9 expression to 40% of wild type at E11.5 and f 51% of wild-type levels in mouse testis at E14.5 as assessed by qRT-PCR. At 11.5 n = 6 XY WT, XY eALDI n = 6 and XX WT n = 3. At E14.5 XY WT n = 6, XY eALDI n = 6 and XX WT n = 4. Error bars are s.e.m. P-values derived from t-tests: *P ≤ 0.05 and ***P < 0.001 compared to wild-type males. Source data are provided as a Source Data file.