Functional analysis of promoter CpG methylation using a CpG-free luciferase reporter vector.
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.
Klug
Maja
M
Department of Hematology and Oncology, University Hospital, Regensburg, Germany.
Rehli
Michael
M
D001483:Base Sequence; D041321:Chromosomes, Human, X; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000172:deficiency / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D018392:Genomic Imprinting; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D015226:Dinucleoside Phosphates; D008156:Luciferases; C015772:CpG dinucleotide