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

<p>Kc cells were transfected with the indicated reporters with or without Axin RNAi, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004509#pgen-1004509-g001" target="_blank">Figure 1</a> and the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004509#s4" target="_blank">Materials and Methods</a>. Sequences of the reporters used are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004509#pgen.1004509.s003" target="_blank">Figure S3</a>. (<b>A</b>) A minR reporter containing two repeats of a 40 bp region of the <i>Tig</i> intron (each repeat contains two WGAWAW and two r-Helper sites) cloned upstream of the <i>hsp70</i> core promoter is sufficient for driving basal expression and mediating Wnt repression. Tig1 and the <i>hsp70</i> core promoter (E.V.) were used as positive and negative controls, respectively. (<b>B</b>) The <i>nkd</i>-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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004509#pgen.1004509.s003" target="_blank">Figure S3</a> for sequence changes). (<b>C</b>) 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. (<b>D</b>) 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).</p>