Fig. 1

Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P < 0.01). d De novo motif analysis results using HOMER. Fold change enrichment of hypermethylated sequences was compared to array background. The top 3 transcription factor motifs included CTCF, BORIS (a CTCF paralogue), and NFκB-p65 (all P < 0.001)