Functional Analysis of Promoter CPG-Methylation using a CpG-Free Luciferase Reporter Vector

Abstract

Methylation of CpG-dinucleotides within proximal promoters is often associated with transcriptional silencing. Methylation-dependent repression is well established for hypermethylated CpG-island promoters that are characterized by a high density of CpG residues. The effect of CpG DNA methylation on CpG-poor promoters is less well characterized, probably due to the lack of convenient assay systems to test promoter activities in vitro. In this report, we describe a novel luciferase reporter vector, pCpGL, which completely lacks CpG dinucleotides and can be used to study the effect of promoter DNA methylation in transfection assays. Whereas a traditional reporter vector that contains a large number of backbone CpG residues significantly represses a CpG-free promoter when methylated, our new reporter vector is only repressed due to the presence of functionally important, methylated CpG residues. The pCpGL vector provides a useful tool to study the effects of CpG-methylation on CpG-rich and CpG-poor promoters.