Figure 3.

BORIS, but not CTCF, DNA binding is methylation independent. A, BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a ³²P-labeled SssI-methylated oligonucleotide containing the H19 CTCF-binding sequence. Lanes 1 and 5, probe alone; lanes 2 to 4 and 6 to 8, incubated with nuclear extract. Supershift assays were done by preincubating extracts with increasing concentrations (+ or ++) of either an anti-BORIS (lanes 3 and 4) or an anti-CTCF antibody (lanes 7 and 8). Arrows, position of the protein-DNA complex and free probe; small arrows, shifted bands. B, BORIS, but not CTCF, DNA binding in vitro is independent on methylation. EMSAs were done with in vitro translated BORIS (left) or CTCF (right) with either an unmethylated oligonucleotide containing a CTCF-binding site (lanes 1, 2, 5, and 6) or an identical SssI-methylated oligonucleotide (lanes 3, 4, 7, and 8). All gel and supershift experiments were done in triplicate. Sections of fluorograms from native gels using a Typhoon phosphorimager are shown. Arrows, the supershift as well as the protein-CTCF-DNA complex and free unbound oligonucleotide probe. C and D, siRNA knockdown of CTCF decreases CTCF and increases BORIS binding to the H19 DMR. HCT116 cells were transfected with either control (Cont)or CTCF siRNA (see Supplementary Fig. S5 for decreased CTCF levels). ChIP was done followed by either (C) QPCR with primers that cover the H19 DMR or (D) MS-ChIP-QPCR via bisulfite treatment and QPCR with either methylated or unmethylated primer sets to the H19 DMR. All ChIP experiments were done in triplicate. Columns, mean; bars, SD. Statistical significance was established by Student's t test. *, P < 0.05.