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. 2015 Apr;27(2):148–155. doi: 10.3978/j.issn.1000-9604.2015.04.03

Figure 3.

Figure 3

Kaiso cannot bind to the hydroxymethylated CGCG-containing CDKN2A sequence in cultured HCT116 cells. (A) Illustration of the strategy to detect methylation and hydroxymethylation in the CGCG-containing DNA sequence in the combined glucosylation-Msp I/Hpa II-PCR assay. PCR products can be successfully amplified from the Msp I-digested hmC-containing DNA sequences after the glucosylation treatment, but not before the treatment. PCR products could be successfully amplified from the Hpa II-digested mC- or hmC-containing DNA sequences before and after the glucosylation treatment; (B) The CDKN2A promoter templates present in the Kasio-IPed chromatin in the CDKN2A-hemimethylated HCT116 cells. The unmethylated-KBS-containing CCND1 templates are also detectable. However, the unmethylated-CGCG-containing GAPDH promoter is not detectable; (C) Hpa II digests only part of CDKN2A templates in the Kaiso-IP chromatin in HCT116 cells, but it digests all of CDKN2A templates in the input control chromatin in the CDKN2A-unmethylated MGC803 cells; (D) The CDKN2A template is amplified in the glucosylated and Msp I-digested input control chromatin. However, the CDKN2A template is not amplified in the glucosylated and Msp I-digested Kaiso-IPed chromatin (bottom image), indicating that Kaiso cannot bind to hmC in the CDKN2A promoter.