Specific DNA/protein interaction is blocked by CpG methylation.

A panel of complementary oligonucleotides comprising CpG 30-mer was synthesized with 5-methylcytosines in all 3 CpGs, in each CpG individually, and in none of the CpGs. Mixing the oligonucleotides creates probes and competitors for electrophoretic mobility shift assays with diverse methylation profiles. (A) Unmethylated CpG 30-mer (U) and CpG 30-mer symmetrically methylated on all 3 CpGs (M) were used as probes and competitors as indicated above panel A. (arrow) Position of major binding complexes. (lanes 10, 11) Probe contains 5-methylcytosine on the sense (S) or antisense (A) strands at CpG1, CpG2, and CpG3. (lanes 6–8) A murine antibody to human aryl hydrocarbon nuclear translocator, ARNT (2B10), was included in the reactions. (open circle) Position of an anti-ARNT supershift. (lane 8) No nuclear extract. (lane 9, asterisk) Addition of a murine antibody to an unrelated antibody. (B) The binding complex from HeLa extracts and unmethylated CpG 30-mer is challenged with a 50-fold excess of double-stranded competitors (see Table). (C) Shifted complexes form with Hep3B extracts and CpG 30-mer symmetrically methylated at either CpG1 (m1), CpG2 (m2), or CpG3 (m3). (lanes 5–15) Shifted complexes formed with Hep3B extracts and unmethylated CpG 30-mer are challenged with CpG 30-mer symmetrically methylated at each CpG (m1-3) or hemimethylated at each CpG on the sense (S1–S3) or antisense (A1–A3) strand. (D) Schematic map of CpG 30-mer.