| Popis: |
We show here that retrovirus and lentivirus vectors are subject to silencing in mouse ES cells producing single-copy proviruses that are silent, variegated or low expressing at best. The mechanism of retrovirus silencing was studied by infecting wild-type and de novo methylase (dnmt3) null ES cells. Silent and variegated retrovirus was identified in both cell types, demonstrating that DNA methylation is not required for establishment of silencing. Wild-type ES cells were also isolated with silent, variegated or low expressing lentivirus. ChIP experiments show that silent and variegated retrovirus and lentivirus are epigenetically marked by the same silent chromatin composed of deacetylated H3 and bound H1, and that H1 binding is further enhanced in dnmt3 null ES cells. In wild-type ES cells, silent and variegated proviruses are DNA methylated, bound by MeCP2 and less H1, and partially reactivated by 5-AzaC but not TSA. These experiments show that DNA methylation plays a partial silencing role but that silent chromatin is the universal mark of retrovirus silencing, and suggest that at least two epigenetic pathways are involved in establishment of silencing. |
