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. 2017 Jun 29;37(14):e00632-16. doi: 10.1128/MCB.00632-16

FIG 3.

FIG 3

Analysis of the role of the Tcf4 binding site in the Sox8 promoter in Wnt-mediated upregulation of Sox8. (A) Genomic architecture of Sox8. The bidirectional promoter of Sox8, its neighboring genes, and locations of the Tcf4 binding site and of the mrhl RNA binding site are depicted. (B) Agarose gel (1.5%) showing the size range of sonicated DNA used for ChIP experiments. (C and D) Tcf4 (C) and β-catenin (β-cat) (D) ChIP, showing their occupancy on the Sox8 gene promoter containing the Tcf4 binding site (bp −800) selectively upon Wnt signaling activation. P7 testes represent the Wnt-repressed state, while P21 testes represent the Wnt-activated state. Ccnd1 (bp −300) is used as a positive control, whereas the β-actin gene (bp −250) is used as a negative control. (E) Occupancy of Tcf4 and β-catenin on the Sox8 gene promoter containing the Tcf4 binding site (bp −800) upon silencing of mrhl RNA. (F) Luciferase assay in Gc1-Spg cells treated with control medium or Wnt3A conditioned medium using plasmid constructs containing the 1-kb upstream promoter region of Sox8 with either the wild-type Tcf4 binding site or a mutant Tcf4 binding site. Data in panels C to F 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.