Molecular Mechanisms of Gene Silencing Mediated by DNA Methylation

Abstract

DNA methylation and chromatin modification operate along a common pathway to repress transcription; accordingly, several experiments demonstrate that the effects of DNA methylation can spread in cis and do not require promoter modification. In order to investigate the molecular details of the inhibitory effect of methylation, we microinjected into Xenopus oocytes a series of constructs containing a human CpG-rich sequence which has been differentially methylated and cloned at different positions relative to a specific promoter. The parameters influencing the diffusion of gene silencing and the importance of histone deacetylation in the spreading effect were analyzed. We demonstrate that a few methylated cytosines can inhibit a flanking promoter but a threshold of modified sites is required to organize a stable, diffusible chromatin structure. Histone deacetylation is the main cause of gene repression only when methylation does not reach levels sufficient to establish this particular structure. Moreover, contrary to the common thought, promoter modification does not lead to the greater repressive effect; the existence of a competition between transactivators and methyl-binding proteins for the establishment of an open conformation justifies the results obtained.DNA methylation is the major modification of eukaryotic genomes and is known to have a profound effect on gene expression. In mammals, this occurs predominantly at the dinucleotide CpG, and approximately 60 to 90% of the dinucleotides are modified (50). In normal cells, methylation involves mainly CpG-poor regions, while CpG-rich areas (CpG islands), located in regulatory regions of class II genes, seem to be protected from the modification (14). This lack of methylation is likely a prerequisite for active transcription; in fact, methylated CpG islands are found on the inactive X chromosome and on silenced alleles of parentally imprinted genes (41,47,48).Genetic experiments have demonstrated that proper control of DNA methylation is essential for normal mammalian development; accordingly, this epigenetic modification seems to play important roles in X chromosome inactivation, genomic imprinting, senescence, and carcinogenesis (3,4,35,36,41,44). The correlation between DNA methylation and gene silencing has been extensively documented by a large body of evidence. In particular, transfection experiments and Xenopus oocyte microinjections, performed with in vitro-methylated DNA, demonstrated that methylation inhibits gene expression (28,29,31,38,56). Conversely, modified silent genes in cultured cell lines can be activated upon treatment with 5-azacytidine, a demethylating agent (18,26).It has been proposed that this modification causes transcriptional repression by directly interfering with the binding of transcription factors to DNA. This hypothesis has been sustained by the identification of a number of transcriptional regulators that cannot bind methylated recognition elements (16). However, the existence of factors indifferent to DNA methylation status a...

Non-linear pharmacodynamics in a non-viral gene delivery system: Positive non-linear relationship between dose and transfection efficiency

“…Methylation of the pDNA can also be an inactivation mechanism and a possible cause of the positive non-linearity observed in the PD [13,14]. Since the saturation of this process by an increased amount of pDNA in the nucleus can enhance transfection activities, methylation could be the cause of the positive non-linearity.…”

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…2,5,28 To clarify the involvement of CpG methylation in lentiviral silencing, we analyzed the methylation of CpG sites in the CMV promoter by bisulfite sequencing method. We analyzed 10 CpG sites close to the 3 0 -end of the CMV promoter from the silenced HR/cHG provirus.…”

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint

“…Evidence is emerging that the methyl-CpG-binding protein 2 (MeCP2) links DNA methylation with histone deacetylases to form a transcriptional repression multiprotein complex. 29,30 The CMV-ie promoter has five palindromic CpG motifs that are potential sites of DNA methylation and promoter silencing. 31 We found that the histone deacetylase inhibitor trichostatin A (TSA) can only partly reactivate the silenced CMV promoter in the synovial tissue ex vivo (manuscript in preparation).…”