FIG. 1.

EMSA analysis of the CTCF site mutations. Radiolabeled oligonucleotide probes derived from the mouse H19/Igf2 ICR (CTCF 1 to 4) or from the chicken β-globin insulator (FII) were incubated with mouse PEF extract. Unlabeled oligonucleotides were included as competitors at 500-fold excess, or anti-CTCF antibody was given to obtain supershifts. (A) Nature of the CTCF site mutations. The DNA sequence spanning CTCF sites 1 to 4 is aligned. CTCF consensus binding sites are marked in bold. The same 10-bp DNA segment wasreplaced in all four CTCF sites by the sequence shown in lowercase letters on top. The NdeI restriction site created to facilitate screening during site-directed mutagenesis is also indicated. (B) Binding of CTCF complexes to the four ICR CTCF sites (left) and competition of CTCF site 3 (right). (C) Competition of CTCF site 2. (D) Lack of gel shift with the mutant CTCF site 2 oligonucleotide. Mutant oligonucleotide was radiolabeled and run next to the wild-type one after incubation with protein extract.