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Fig. 3 | Clinical Epigenetics

Fig. 3

From: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

Fig. 3

DNA methylation alterations occur at CTCF binding sites after CTCF knockdown in vitro and methylation inhibition reintroduces gene expression. Stable E6/E7 cell lines expressing CTCF shRNAs were cultured up to 10 days. a Validation of LTBP2 transcriptional silencing after 10 days of shCTCF induction by qPCR. Data are shown mean ± SD of technical triplicates from one representative experiment of three. b ChIP-qPCR for CTCF at LTBP2 promoter associated CTCF binding site (pCBS) ~ 400 bp upstream of LTBP2 transcription start site. Showing a reduction in CTCF binding after 10 days of shCTCF induction. Data shown are mean + SD of technical triplicates from one representative experiment of three. *P < 0.05 and **P < 0.01. c Pyrosequencing of bisulfite DNA demonstrating increased methylation at LTBP2 promoter CTCF binding site after 10 days of shCTCF1 induction. Data shown are mean ± SD of technical triplicates from one representative experiment of three. shCTCF2 induction and controls are shown in Supp Fig S4. d Decreased TNFAIP3, FGF5, EPHA3, and AMIGO2 transcriptional silencing after 10 days of CTCF knockdown. At day 5, Dox + cells were also exposed to 5-aza-2 deoxycytidine a methyltransferase inhibitor at a low 0.2 uM dose that does not result in significant growth inhibition. Data shown are mean ± SD of technical triplicates from one representative experiment of three. *P < 0.05 and **P < 0.01. e ChIP-qPCR for CTCF demonstrating decreased binding activity at promoter associated CTCF binding sites of candidate genes after 10 days of CTCF knockdown (for controls and expanded results see Supp Fig S3). f MeDIP-qPCR of promoter associated CTCF binding sites exhibiting loss of CTCF binding. Methylation increases were detected accompanying reduced CTCF binding. g Methylation B-values and mRNA (log2 RSEM) expression levels compared for LTBP2 gene in TCGA prostate tumors (Cell 2015). Pearson correlation R-value shown. Data was downloaded from cBioPortal for PRAD TCGA samples (Cell 2015). Decreased mRNA expression correlates with greater LTBP2 methylation

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