Fig. 7: CHD6 transcriptionally regulates TMEM65 by cooperating with TCF4.

a HCT116 cell lysates were immunoprecipitated with an anti-CHD6 antibody and were immunoblotted with the indicated antibodies. b Representative immunoblot analysis of fractions containing protein complex eluted from size exclusion chromatography. Cell lysate extracted from CHD6-transfected HEK293T cells was analyzed for CHD6-containing complexes using Superose 6 (GE) gel filtration column. Eluted fractions of protein complex were collected and revealed by immunoblotting with the indicated antibodies. CHD6, TCF4, and β-catenin were co-eluted at fractions 29–32. c TCF4 binding motif obtained from JASPAR (top). The potential binding site of TCF4 on the promoter of TMEM65 (bottom). d ChIP of CHD6 and IgG in control or CHD6 KD HCT116 cells, followed by qPCR for the loci identified by searching for binding sites on TMEM65 promoter. e ChIP of TCF4 and IgG in control and CHD6 KD HCT116 cells followed by qPCR for the loci of predicted binding sites on TMEM65 promoter. f RT-qPCR analysis of TMEM65 mRNA in HCT116 cells treated with Wnt pathway inhibitors IWR-1-endo (20 μM), XAV-939 (1 μM), or vehicle. g RT-qPCR analysis of TMEM65 mRNA in HCT116 cells cultured with L-Wnt3a-expressing cell CM. h RT-qPCR analysis of TMEM65 mRNA in HCT116 cells cultured with L-Wnt3a-expressing cell CM, in the presence or absence of Wnt pathway inhibitor IWR-1-endo (20 μM). i TMEM65-reporter luciferase activity in 293T cells with or without CHD6 overexpression. j TMEM65-reporter luciferase activity in 293T cells with overexpression of β-catenin and TCF4 alone, or in combination. k TCF4 binding motif obtained from JASPAR (top). The potential binding site of TCF4 on the promoter of CHD6 (bottom). l ChIP of TCF4 and IgG in control or CHD6 KD HCT116 cells, followed by qPCR for the loci on CHD6 promoter. m Immunoblot and RT-qPCR analysis of CHD6 in HCT116 cells treated with Wnt pathway inhibitors LGK974. n Immunoblot and RT-qPCR analysis of CHD6 in HCT116 cells cultured with L-Wnt3a-expressing cell CM. o Immunoblot and RT-qPCR analysis of CHD6 in HCT116 cells cultured with L-Wnt3a-expressing cell CM, in the presence or absence of Wnt pathway inhibitors LGK974 (10 μM). p ChIP of β-catenin and IgG in HCT116 cells, followed by qPCR for the loci on CHD6 promoter. Assays were performed with three replicates. P values were calculated by two-tailed t-test (d, e, i, l, p) or one-way ANOVA (f–h, j, m–o). Data are presented as means ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.