Abstract
The human cytomegalovirus (CMV) immediate early promoter has been extensively used to drive target gene expression in transgenic mammalian cells. DNA methylation of the CMV promoter has been shown to be the reason for a reduced promoter activity and silencing of the target gene. We have established an in vitro model system, in which human brain cancer cells (glioblastoma multiforme, GBM) were transfected with pAdTrack-CMV-GFP plasmid, isolated from a dcm positive (dcm+) E. coli strain. We found that in two CCTGG sequences located at position from -304 to -300 nt and from -497 to -493 nt of the CMV promoter region, the internal C was methylated in all analyzed clones, i.e., the E. coli dcm methylation pattern is maintained in the CMV promoter region after its integration into the human genome. In contrast, we found that the recognition sites for the transcription factor NFkB and certain other transcription factors in the enhancer region of the CMV promoter (from -107 to -270 nt) were hypomethylated. This might explain why the CMV promoter maintained an active mode, driving the GFP expression despite the demonstrated methylation of the CMV promoter. We noticed that the CCTGG sequence is also contained in the binding sequence motif of transcription factor NFkB. Hence we have comprehensively studied transcription factors through a database searching, and the responsive elements that contain dcm methylation sequences CCW(A/T)GG. A list of transcription factors and the corresponding regulated genes are presented.
Highlights
DNA methylation has attracted great interest due to its important role in regulation of the gene expression in most eukaryotes [1,2,3]
We demonstrate that when a plasmid, containing the immediate early CMV promoter isolated from E. coli, is transfected into human GBM cells, the E. coli dcm methylation with the two CCTGG sequences in the CMV promoter region is maintained after stable integration in the human genome
The human cytomegalovirus (CMV) immediate early gene promoter has been extensively used in transfection systems to drive target gene expressions in mammalian cells
Summary
DNA methylation has attracted great interest due to its important role in regulation of the gene expression in most eukaryotes [1,2,3]. DNA methylation in eukaryotes mainly occurs in cytosine residues in palindromic CpG dinucleotide. In vertebrates such CpG sites are not evenly distributed in the genome but are present in only one fifth of its predicted random frequency. Evidence obtained from the study of DNA methylation in eukaryotes indicates that active gene expression as a rule requires unmethylated CpG sites especially in the promoter region. Regional hypermethylation events occurred accompanying a global hypomethylation. Loss of gene expression is frequently caused by hypermethylation of the promoter region of the gene, such as tumor suppressor genes [12,13,14]. Analysis of DNA methylation has a potential impact on cancer risk assessment, chemoprevention and gene therapy
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