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. 2017 Aug 2;7:7116. doi: 10.1038/s41598-017-07559-0

Figure 6.

Figure 6

Kaiso binds to the promoters of BCL2 and BAX, at least partly, via methylated DNA. HUVECs (A) and HMEC-1s (B) were treated with 5 μM 5-azacytidine for 72 h. DNA fragments (200–500 bp) were obtained from nuclear lysates and were subjected to chromatin immunoprecipitation. Mouse monoclonal anti-Kaiso 6F/6F8-CHIP grade antibody was used for Kaiso/DNA complex precipitation and the precipitated DNA fragments were amplified using primers 10–13 (BCL2) (A,a and B,a), primer 2 (BAX) (A,b and B,b) and primer 7–10 (BIK) (A,c and B,c). Primer positions (horizontal arrows) are shown in the schematic drawings in the upper panel in A and B. Asterisks mark the positively amplified promoter regions. Normal mouse IgG was used as a negative control. M: DNA marker. (A) In HUVECs, after 5-azacytidine mediated DNA demethylation, Kaiso remained binding to the promoter regions −601 to −1 bp of BCL2 and −1201 to −1 bp of BIK (Asterisks) while no longer binding to the promoter region −645 to −316 bp of BAX. (B) In HMEC-1s, after 5-azacytidine mediated DNA demethylation, Kaiso remained binding to the promoter region −601 to −1 bp of BIK (Asterisks) while no longer binding to the promoter regions −1201 to −1 bp of BCL2 and −645 to −316 bp of BAX. DNA methylation/demethylation in BAX promoter region −661 to −301 bp was determined using methylation specific PCR (Supplementary Figure S5).