Methylation of CHD5 promoter region down-regulates CHD5 expression in breast cancer. (A) Methylation status of CHD5 promoter region in nine breast cancer cell lines as determined by bisulfite sequencing of cloned PCR products. The horizontal line at the top represents promoter DNA, whereas vertical lines under it indicate the relative locations of each CpG site. Methylation status of each CpG site is indicated by circles, and dark, gray and empty circles indicate high levels (methylation frequency > 50%), moderate levels (methylation frequency < 50%), and no (methylation frequency = 0) methylation, respectively. (B, C) Detection of CHD5 mRNA levels by real time PCR in Hs 578T and MDA-MB-231 cell lines treated with 2 and 5 μM 5-aza-2'-deoxycytidine (AZA) for 3 days (B) or in MDA-MB-231 cells treated with 5 μM AZA for 12 and 24 hours (h) (C). GAPDH serves as an internal control. (D) Methylation levels of CHD5 promoter in MDA-MB-231 cells treated with AZA, as detected by bisulfite treatment and sequencing of five clones from each PCR product. Empty, gray and dark circles indicate zero, one to three and four or five clones, respectively, that are methylated. (E) Methylation levels of CHD5 promoter in breast tumors (n = 30) and normal breast tissues (n = 10). The frequency of methylation (y axis) at each CpG site (x axis) was the average frequency in either the tumor or the normal group.