Swapping HMG and C-clamp binding sites switches the transcriptional output of W-CRMs.

Kc cells were transfected with the indicated reporters with or without Axin RNAi, as described in Figure 1 and the Materials and Methods. Sequences of the reporters used are listed in Figure S3. (A) A minR reporter containing two repeats of a 40 bp region of the Tig intron (each repeat contains two WGAWAW and two r-Helper sites) cloned upstream of the hsp70 core promoter is sufficient for driving basal expression and mediating Wnt repression. Tig1 and the hsp70 core promoter (E.V.) were used as positive and negative controls, respectively. (B) The nkd-IntE W-CRM reporter, which is activated by Wnt signaling, is converted to a repressed W-CRM when its three functional HMG sites and two Helper sites were replaced by five WGAWAW and r-Helper pairs (see Figure S3 for sequence changes). (C) The Tig1 W-CRM reporter is activated by Wnt signaling when two WGAWAW sites and two r-Helper sites were converted into classic HMG-Helper pairs. (D) The switch of the Tig1 W-CRM to an activated W-CRM requires swapping both WGAWAW and r-Helper sites. When one motif is swapped without the other, low basal activity and little activation was observed. *p<0.05; **p<0.01; n.s.: not significant (Student's T-test).