Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation

The majority of 5-methyllcytosine in mammalian DNA resides in endogenous tr ansposable elements and is associated with the transcriptional silencing of these parasitic elements. Methylation also plays an important role in the silencing of exogenous retroviruses. One of the difficulties inherent in th e study of proviral silencing is that the sites in which proviruses randoml y integrate influence the probability of de novo methylation and expression . In order to compare methylated and unmethylated proviruses at the same ge nomic site, we used a recombinase-based targeting approach to introduce an in vitro methylated or unmethylated Moloney murine leukemia-based provirus in MEL cells. The methylated and unmethylated states are maintained in vivo , with the exception of the initially methylated proviral enhancer, which b ecomes demethylated in vivo. Although the enhancer is unmethylated and remo deled, the methylated provirus is transcriptionally silent. To further anal yze the repressed state, histone acetylation status was determined by chrom atin immunoprecipitation (ChIP) analyses, which revealed that localized his tone H3 but not histone H4 hyperacetylation is inversely correlated with pr oviral methylation density. Since members of the methyl-CpG binding domain (MBD) family of proteins recruit histone deacetylase activity, these protei ns may play a role in proviral repression. Interestingly, only MBD3 and MeC P2 are expressed in MEL cells. ChIPs with antibodies specific for these pro teins revealed that only MeCP2 associates with the provirus in a methylatio n-dependent manner. Taken together, our results suggest that MeCP2 recruitm ent to a methylated provirus is sufficient for transcriptional silencing, d espite the presence of a remodeled enhancer.