Figure 6

The formation of unusual DNA structure by CTCF is not dependent on DNA sequence composition downstream of CTCF binding site. (A) Construction of probes: the pBEND2-FII-forward or reverse plasmids (lines 1 and 6) were digested with HindIII and SalI to remove DNA sequence to the right of the FII insulator element. This sequence was replaced with a shuffled version of the original sequence (black line, lines 2 and 7) or random DNA sequence (grey line, lines 3 and 8). Lines 4 and 5 show control probes containing original FII site and downstream DNA sequence digested with MluI or BamHI. (B) Mobility shift assays were performed by incubating CTCF with radiolabeled 173 bp probes generated by HindIII and BamHI double restriction digests of pBEND2-FII-reverse-random, pBEND2-FII-reverse-shuffled, pBEND2-FII-forward-random or pBEND2-FII-forward-shuffled. In lane 1, the control MluI digested pBEND2-FII-Forward probe shows the rapidly migrating CTCF-probe complexes. Lanes 2 and 3 show the FII-reverse shuffled and random probe complexes have a similar mobility to the control MluI complexes in lane 1. Lanes 4 and 5 show that the FII-forward shuffled and random probe complexes have a similar mobility to the control BamHI probe in lane 6. Therefore, DNA sequence downstream of FII binding site does not affect CTCF's ability of to form a DNA loop. All samples were run on the same polyacrylamide gel, although the lanes were re-arranged electronically.