Figure 1: GCM1 is a direct transcriptional target for β-catenin/BCL9L/TCF4 signalling.

(a) BeWo cells transfected with the indicated siRNAs were treated with 50 μM FK or vehicle (control) for 48 h and assayed for TOP or FOP activities. siβ-cat-1, -2 denote siRNAs targeting different sites of β-catenin mRNA. (b) qRT–PCR analysis of the expression levels of Wnt target genes in FK-treated BeWo cells. (c) Portions of nuclear lysates prepared for ChIP assays were analysed by immunoblotting. The closed and open triangles indicate 80 kDa (modified) and 60 kDa TCF4s, respectively. Representative data from three independent experiments are shown. (d) Schematic representation of the genome structure of GCM1. Open and closed triangles indicate TBEs and a negative control region containing no TBEs within a region 1 kb up- or downstream, respectively. Ex, Exon; Int, Intron. (e,f) ChIP assays with anti-β-catenin antibody. GAPDH and AXIN2 are negative and positive controls, respectively. (g) Schematic representation of the luc-Int2-TBE10 and luc-Int2-TBE10-mut reporter constructs for enhancer assays. (h) Cis-activation potential of the region containing TBE10 or its mutant in response to β-catenin/BCL9L/TCF4 signalling was evaluated by luciferase enhancer assays. Values are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. N=3 (a,b,e) or N=4 (f,h). Statistical tests were performed using unpaired two-tailed Student's t-test.