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.

Region-specific alteration of histone modification by LSD1 inhibitor conjugated with pyrrole-imidazole polyamide

“…Py-Im polyamides have affinities for specific DNA sequences as strong as transcription factors, and these molecules can interfere with the binding of transcription factors to their recognition sites [ 26 , 29 , 31 ]. Applying the principles of these studies, we and Dervan's group recently demonstrated that Py-Im polyamides recognizing DNA with CpG sequences inhibited the induction of DNA methylation in vitro and in cellulo [ 35 , 36 ]. While these studies showed that Py-Im polyamides could be utilized to inhibit DNA methylation in a region-selective manner, the present study suggested that Py-Im polyamides could be also utilized for region-selective alteration of histone modification by conjugation with epigenetic inhibitors.…”

“…Similar to our study, Sugiyama and colleagues conjugated a library of Py-Im polyamides with HDAC inhibitors or histone acetyl transferase activators and reported that each conjugate shows histone acetylation and gene activation in living cells in a different group of genes [ 37 − 39 ]. Because these conjugates are small molecules, they can easily be taken up through the cell membrane and localized to the nuclei [ 28 , 36 , 40 ]. In these previous reports, however, the link between epigenetically altered regions and sequences recognized by each Py-Im polyamide was not necessarily clear.…”

Implications for the colorectal surgeon following the 100 000 Genomes Project

“…Epigenetic changes are potentially reversible, and many drugs, such as DNA methyltransferase inhibitors, have been developed that target these changes. Unfortunately, there have been disappointing results in clinical trials, probably due to the complexity of the mechanisms underlying epigenetic change [4]. As knowledge of cancer biology improves new drug targets are being identified and developed to provide more personalized treatments.…”

Spicing Up an Interdisciplinary Chemical Biology Course with the Authentic Big Picture of Epigenetic Research

“…In the past decade, a handful of small molecules have accessed and targeted the nucleosomal DNA successfully. , Particularly, the sequence-specific DNA binders are examples: pyrrole-imidazole polyamides interact with the nucleosome , and can control epigenetic processes . Apart from inhibition of DNA methylation which causes major epigenetic changes, polyamide hairpins have been used as nucleosomal clamps connecting two aligned minor grooves. They have further been used as cyanine dye–polyamide conjugates in fluorescence studies, such as Förster resonance energy transfer (FRET) on the nucleosome .…”