Bipartite Recognition of DNA by TCF/Pangolin Is Remarkably Flexible and Contributes to Transcriptional Responsiveness and Tissue Specificity of Wingless Signaling
Figure 3
HMG-Helper pair configuration preferences in cell culture reporter assays.
Kc cells were transfected with the indicated reporters with or without a plasmid expressing Arm* and assayed for luciferase expression. (A) Synthetic W-CRMs containing two HMG-Helper site pairs in all four orientations with 0, 3, 6, 9 or 12 bp spacing between the HMG and Helper site (see Table S1 for sequences of each synthetic W-CRM). In every construct, the two pairs were separated by 6 bp. Two constructs containing only HMG sites (HMG1 and HMG2) were included in the analysis and the reporter vector without an insert is referred to as empty vector (EV). The data shown represents the average of three independent experiments with three biological replicates each. Error bars represent SD and asterisks indicate a significant difference in activation compared to HMG site constructs. (B) The cartoon at the top of the panel depicts the nkdIntE2P2 W-CRM, all HMG and Helpers sites were mutated (striped arrowheads) except one AK6 pair (green box). The spacing and orientation of this pair were altered as indicated. The endogenous AK6 configuration displayed the highest level of activation by Arm*, while AK0, AK12 and FF0 constructs also exhibited higher activation than the HMG site only control. The data are the means of two independent experiments with three biological replicates each, ± SD. Asterisks indicate a significant increase in activation compared to the HMG site only construct. (C) In the nkdUPE2 W-CRM, the HMG and Helper site pair (green box) which contributes most potently to activation [57] was altered as indicated. AK1 and AK6 configurations responded as well as the endogenous FF1 pair, while the FF6 configuration was less active. The data are the means of three biological replicates ± SD, and are representative of several independent experiments. Asterisks indicate reduced activation by Arm* compared to the wild-type construct. In all cases, Student's T-tests were employed to determine significance.