FIG 5.

c-Myc binds to the Sox8 promoter at the Myc-Max-Mad binding site upon Wnt signaling activation. (A) The architecture of the 1-kb upstream promoter of the Sox8 gene with the CAAT box, Tcf4 binding site, mrhl RNA binding site, and putative transcription factors is identified along with the binding sites. (B) Expression analysis of c-Myc, Max, and Mad in Gc1-Spg cells (control, Wnt3A conditioned medium treated). (C) c-Myc ChIP in Gc1-Spg cells and mouse testis with Cad as a positive control for c-Myc occupancy. The region from bp −500 to −700 in the Sox8 promoter was used as a negative control. (D) Expression analysis of c-Myc and Sox8 upon c-Myc silencing in control and Wnt3A conditioned medium-treated Gc1-Spg. (E) ChIP showing recruitment of c-Myc-associated coactivator Pcaf at the Myc-Max-Mad binding site in Wnt3A conditioned medium-treated Gc1-Spg cells and in P21 mouse testes. (F) Increased level of H3K9ac at the Myc-Max-Mad binding site upon Wnt activation in Gc1-Spg cells and in P21 mouse testes. Data are plotted as means ± SD, n = 4. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 (two-tailed Student t test). N. S, not significant.