K4-dependent promoters are DNA hypermethylated. (a-c) Percentage DNA methylation of indicated K4-dependent (K4-D) and K4-independent (K4-I) genes in SW480 (a), V432 (b), and V441 (c). For comparison, methylation levels of genes were also quantified in normal colon crypts. Error bars indicate the standard deviation of pyrosequencing assays performed on three independent preparations of normal colon crypts. All K4-independent genes analyzed contain a CpG island at the TSS. All K4-dependent genes, except PTGDR, lack a CpG island at the TSS. (d) Quantitative RT-PCR analyses of genes from 5-azacytidine (Aza) treated (+) and untreated (-) SW480 cells. Data were normalized to GAPDH. RHBDL2, UBA7, FUT3, and GUCY2C are K4-dependent genes that lack a CpG island. ZFP42 was previously found in this cell line to be DNA hypermethylated and reversible upon treatment with 5-azacytidine, and thus serves as a positive control. OVOL1 is a K4-independent gene with a promoter-associated CpG island and serves as a negative control. Error bars indicate the standard deviation of quantitative RT-PCR reactions performed in triplicate. *P ≤ 0.01.