Fig. 2

Analysis of Sox5 binding to the dmrt1bY promoter and regulation of dmrt1bY promoter activity upon modulation of Sox5 expression. a Chromatin immunoprecipitation (ChIP) of Sox5 binding to regions α, β, or γ of the dmrt1bY proximal promoter. Transient transfection of a flagged and tagged version of Sox5 into either medaka spermatogonial or fibroblast cell lines and subsequent immunoprecipitation (FLAG antibody) followed by the quantitative real-time polymerase chain reaction. Results are presented as enrichment compared to the input and correspond to three independent immunoprecipitations for each region (α, β, or γ). Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 3). b1–b4 Analysis of dmrt1bY proximal promoter activity after Sox5 transient transfection into the medaka spermatogonial cell line (Sg3). Deletions of the 5′ dmrt1bY promoter were generated: b1 α region, b2 α and β regions, b3, b4 α, β, and γ regions. Transcriptional activity was quantified in the absence (control, -Sox5) or presence (+Sox5) of Sox5. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 4). c Detailed analysis of the transcriptional activity of the alpha (α), alpha-mutant (α-MUT), and beta (β) fragments. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 4). * p value ≤ 0.05, ** p value ≤ 0.01. ns non-significant, OLF Oryzias latipes fibroblast, Sg3 Oryzias latipes spermatogonial cell