Inhibition of DNA Methylation at the MLH1 Promoter Region Using Pyrrole-Imidazole Polyamide

Abstract

Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2'-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)-imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_-16 (Py-Im-β-Im-Im-Py-γ-Im-Py-β-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human MLH1 gene. MLH1 is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_-16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the MLH1 promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_-16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the MLH1 promoter under hypoxic conditions (1% O₂), methylation of the MLH1 promoter was inhibited in the region surrounding the compound binding site. Silencing of the MLH1 expression was also inhibited. Promoter methylation and silencing of MLH1 were not inhibited when MLH1_non was added. These results indicate that Py-Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells.

Figure 1

Chemical properties of hairpin PIPs targeting the MLH1 promoter. (A) Chemical structure of MLH1_–16 and MLH1_non. (B) Schematic diagram showing the binding site of MLH1_–16 near the TSS (arrowhead) of the human MLH1 promoter. The expected binding site is indicated with an arrow and in red. There is no binding site of MLH1_non within 1 kb from the TSS of MLH1. Ball-and-stick models of the polyamides are shown between the two DNA strands. Closed and open circles represent Im and Py rings, respectively, diamonds represent β-alanine, and curved lines represent γ-aminobutyric acid. (C) Investigation of binding affinities of MLH1_–16 and MLH1_non to target DNA in vitro by EMSA on a 20% polyacrylamide gel. Lane 1, double-stranded untreated DNA; lane 2, DNA treated with 5% DMSO (control); lanes 3–7, DNA treated with various concentrations of MLH1_–16; and lanes 8–12, DNA treated with various concentrations of MLH1_non. Mobility of 5′-FAM-labeled DNA corresponding to the MLH1_–16 binding site (red letters) was delayed in the presence of MLH1_–16 (5 or 10 μM), but not in the presence of MLH1_non.

Figure 2

Site-specific inhibition of DNA methylation by MLH1_–16 in vitro. (A) Schematic presentation of plasmid DNA including the promoter region of MLH1 (−780 to +483, white bar) that was used in this assay. MLH1_–16 binding site is indicated with a black triangle. Eight CpG sites at positions −70, −63, −27, −16, −1, +156, and +171 are shown as red, pink, light gray, gray, black, light blue, and blue bars, respectively. (B) Pyrosequencing analysis of DNA methylation levels in the MLH1 promoter following DNA methylation and treatment with MLH1_–16 or MLH1_non in vitro. Colored bars match those in (A). Data represent the mean of three independent replicates ±SD. DNA methylation was inhibited at positions −27, −16, and −1 in the presence of MLH1_–16.

Figure 3

Distribution of FAM-labeled MLH1_–16 in RKO cells after 1 day of incubation. (A) Chemical structure of the FAM-labeled MLH1_–16. (B) Cells were visualized using fluorescence microscopy following nuclear staining with DAPI (blue or red). MLH1_–16 was localized in the nuclei of RKO cells after 1 day. MLH1_non was also confirmed to be localized in the nuclei (Figure S1).

Figure 4

DNA methylation levels of MLH1 in RKO cells treated with MLH1_–16 or MLH1_non for 30 days. RKO cells were treated with 0.1% DMSO (control), 5 μΜ MLH1_–16, or 5 μΜ MLH1_non under normal conditions (20% O₂). Data represent the mean of three independent replicates ±SD. No significant reduction of DNA methylation at positions −27, −16, and −1 was observed compared with untreated control (day 0).

Figure 5

Decreased DNA methylation and increased MLH1 mRNA expression induced by AZA or AZA + TSA treatment of RKO cells. (A) DNA methylation levels in the MLH1 promoter of RKO cells treated with DMSO (control), AZA, or AZA + TSA. Data represent the mean of three independent replicates ±SD. The methylation level in the MLH1 promoter was decreased by AZA or AZA + TSA treatment. (B) RT-PCR analysis of the MLH1 expression in RKO cells treated with DMSO (control), AZA, or AZA + TSA. GAPDH was used as an internal control. MLH1 was re-expressed somewhat or markedly by treatment with AZA or AZA + TSA, respectively.

Figure 6

Increased DNA methylation and decreased MLH1 mRNA expression in RKO cells incubated under hypoxia. (A) DNA methylation levels in the MLH1 promoter of RKO cells treated with AZA or AZA + TSA and then incubated under hypoxic conditions (1% O₂) for 0, 20, or 30 days (H0, H20, H30, respectively). Data represent the mean of three independent replicates ±SD. The methylation level of the MLH1 promoter was increased under hypoxic conditions. (B) RT-PCR analysis of the MLH1 expression in RKO cells treated with AZA or AZA + TSA and incubated under hypoxic conditions for 0, 20, or 30 days. GAPDH was used as an internal control. MLH expression was silenced under hypoxic conditions.

Figure 7

Selective inhibition of DNA methylation and MLH1 silencing by MLH1_–16 in RKO cells incubated under hypoxia. (A) DNA methylation levels in the MLH1 promoter of RKO cells treated with AZA + TSA and then incubated under hypoxic conditions (1% O₂) with DMSO (control), 5 μΜ MLH1_–16, or 5 μΜ MLH1_non. Data represent the mean of three independent replicates ±SD. **P < 0.01, N.S. not significant (P > 0.05). Increase of DNA methylation at positions −70, −63, −27, −16, −1, +156, and +171 was inhibited in the presence of MLH1_–16, but not in the presence of MLH1_non. (B) RT-PCR analysis of the MLH1 expression in RKO cells treated with AZA + TSA and then incubated under hypoxia with DMSO (control), MLH1_–16, or MLH1_non. GAPDH was used as an internal control. Silencing of MLH1 was inhibited in the presence of MLH1_–16.