Figure 1

CTCF forms unusual DNA structure at β-globin FII insulator element. (A) Diagram of pBEND2 vector containing the FII insulator in "forward" orientation (rightward-pointing arrowhead). Restriction digestion generates 166 bp probes with FII site permuted from right (M) to left end of fragment (Ba). (B) CTCF, + or -SUMO1, was incubated with ³²P-labelled probes. The CTCF translation product contains mixture of both full-length and C-terminally truncated protein; each exhibits same mobility pattern. (C) Expected behaviour of fragments in DNA bending assay. Mobility of protein-DNA complex decreases as DNA binding site is permuted towards centre of probe and increases as site is permuted towards ends of probe. Below schematic of electrophoretic mobilities of permuted fragments is a diagram of the best fit curve of relative mobilities (μ) as function of position (bp) from left EcoRV site to enzyme site used to generate the probe. Dotted lines indicate that bend centre is at position 80 bp. (D) Schematic of an asymmetrical DNA bend. The electrophoretic mobilities of permuted fragments plotted as in (C). The shape of best fit curve resembles that for a symmetrical DNA bend except bend centre is 90 bp. (E) Relative mobilities of CTCF-probe complexes plotted and fitted as described above. Note unusual shape of the curve compared to that in Figure 1C. SUMOylated and unmodified full length CTCF (top complex, bottom two curves) are different indicating efficient SUMOylation of CTCF in vitro. Top two curves (representing the bottom complexes) are similar.