Figure 5. Experimental validation of predicted cooperative DNA binding by three TF pairs, ZIF with DLL, GT with TTK and D with MAD.

Relative recovery of luciferase-tagged TF with a biotinylated target DNA sequence is measured in the presence of one or both TFs and various unlabeled competitor sequences. (A) Examples of wild type and mutant competitor sequences are shown for the analysis of ZIF with DLL. The sequences for all competitor sequences are shown in Table S16. The wild type sequence has a strong predicted TF binding sites, shown in bold type, for the luc-tagged TF (ZIF, GT, D) and for the hypothesized interacting TF (DLL, TTK, MAD respectively). As controls, competitor sequences are used where either one (ΔZIF or ΔDLL) or both (ΔZIFΔDLL) TF binding sites are disrupted or the spacing between sites has been increased by 5 bp (+5). An additional competition experiment uses two competitor DNAs (ΔZIF + ΔDLL), each of which is at the same concentration as the single competitor DNAs in the other samples. Altered or inserted nucleotides are shown in red. Genomic sequences flanking the binding sites are in grey. (B) The luciferase activity recovered bound to the biotinylated wild type probe was measured in the presence of different competitors listed on the X-axis. A dash is used to indicate no added competitor DNA. Luciferase measurements are reported relative to a sample using the wild type sequence as a competitor (Y-axis). In the upper panels, recovery of the luciferase-tagged protein is measured in the presence of the hypothesized interacting TF present as an MBP tagged protein. In the presence of the secondary TF, wild type sequences compete better for binding than sequences in which the sequence of or spacing between predicted binding sites is disrupted. In the lower panels, recovery is shown in the absence of the second protein. For all three primary TFs, little activity is recovered in the absence of the secondary TF, regardless of the competitor DNA.