Figure 3

CTCF forms the unusual DNA structure at the c-myc P2 promoter and at the chicken F1 lysozyme gene silencing element. (A) The vector pBEND2-c-myc P2 was digested with MluI (M), BglII (Bg), NheI (N), SpeI (Sp), EcoRV (E), SmaI (Sm), KpnI (K) and BamHI (Ba) to make a series of permuted 292 bp probes The c-myc P2 promoter region was inserted into two XbaI sites (X). The ³²P-labelled, permuted probes were incubated with SUMOylated CTCF lanes 10-17 and unmodified CTCF lanes 1, 3-9 (lane 2 is empty). The reactions were carried out as in Figure 1B. (B) The vector pBEND2-F1, containing the chicken lysozyme gene F1 silencing element inserted into two XbaI sites, was digested with MluI (M), BglII (Bg), NheI (N), SpeI (Sp), EcoRV (E), SmaI (Sm), KpnI (K) and BamHI (Ba) to make permuted ³²P-labelled 178 bp probes. CTCF was made by in vitro transcription/translation and used directly in electrophoretic mobility shift assays with the permuted probes.