Figure 4

SOX2 directly binds to the DKK1 promoter region. (a) Sequence homology of region around DKK1 between mammals. Except for the exons of DKK1, the region 1 kbps upstream of the transcriptional start site of DKK1 was highly conserved and contained an HMG homeodomain (−76 bps). (b) ChIP assay for the −76 bps region of DKK1 showed SOX2-specific binding. (c) Luciferase reporter assay. Luciferase activity and transfection efficiency were normalized to transfection of the SV40 promoter control vector (SV40 promoter). The human DKK1 promoter region from −974 bps to +119 bps around the DKK1 transcription start site, which contains an HMG-binding domain, was cloned into the luciferase enhancer vector (DKK1 promoter). (d) The luciferase activity of sh-SOX2 was significantly decreased compared with that of the sh-control. ^***P<0.001. (e) Site-directed mutagenesis, involving single (M1) and triple (M3) mutations, was performed in the HMG domain of the DKK1 promoter vector. (f and g) The M1 (also SOX2 binding site) mutation had no effect on the luciferase activity, but the M3 mutation significantly reduced the luciferase activity in both hUCB–MSCs and tera-1 cells. ^***P<0.001