Figure 6.

WNT8B Is a Major Downstream Target of SOX21

(A) Representative western blot analysis of regional markers and Wnt signaling proteins in WT, SOX21^−/−, WNT8B^−/−, and SOX21^−/−WNT8B^−/− NPCs at day 6. WT, wild type; SOX21 and WNT8B double KO clones: S13W5 (SOX21^−/− no. 13, WNT8B^−/− no. 5), S13W13 (SOX21^−/− no. 13, WNT8B^−/− no. 13), S23W1 (SOX21^−/− no. 23, WNT8B^−/− no. 1).

(B) Examination of Wnt signaling activity by TOP/FOPFlash reporter assays in WT, SOX21^−/−, WNT8B^−/−, and SOX21^−/−WNT8B^−/− NPCs at day 5. Data are shown as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (n = 4).

(C) ChIP-qPCR results of SOX21 at the WNT8B locus in NPCs at day 5. Data are shown as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 (n = 3). NegCtrl stands for the negative control, a gene desert region (chr21:25509072 + 25509220, GRCh37/hg19).

(D) Representative coimmunoprecipitation results for interactions between endogenous SOX21 and β-catenin in WT and SOX21 KO NPCs at day 7. IP: immunoprecipitation; WB: western blot.

(E) ChIP-seq signal profiles of SOX21 at the WNT8B locus in WT NPCs at day 5. ChIP-seq signal profiles of TCF4 in hESCs and anterior neural ectoderm cells (ANECs) were obtained from published data. The gray box above the signal tracks indicates the SOX21 binding site at the WNT8B locus.

(F) ChIP-qPCR analysis of TCF4 and β-catenin binding at the enhancer of WNT8B in WT and SOX21 KO NPCs at day 5. Data are shown as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 (n = 3).

(G) Representative coimmunoprecipitation results of interactions between endogenous TCF4 and β-catenin in WT and SOX21 KO NPCs at day 7. IP: immunoprecipitation; WB: western blot.

(H) Results of luciferase reporter assays using an empty vector containing only a minimal promoter (minP) and vectors containing either the WT WNT8B enhancer sequence or the enhancer sequence with mutated TCF4 binding sites (WNT8B Mut), respectively, in response to Wnt signaling activation by CT99021 (0.4 μM) at day 6. Mutations at the TCF4 binding sites are indicated by red letters. Data are shown as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (n = 4).

(I) Examination of WNT8B enhancer luciferase reporter activity in WT and SOX21 KO NPCs at day 6. Data are represented as ratios of luciferase activity relative to that with an empty vector containing a minP only. Data are shown as means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (n = 4).

See also Figure S5.