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Retroviral and adeno-associated viral sequences can dramatically silence transgene expression in mice. We now report that this repression also occurs in stably infected HeLa cells when the cells are grown without selection. Expression of a transduced lacZ gene (rAAV͞CMVlacZ) is silenced in greater than 90% of cells after 60 days in culture. Surprisingly, high-level expression can be reactivated by treating the cells with sodium butyrate or trichostatin A but not with 5-azacytidine. When cell clones with integrated copies of rAAV͞CMVlacZ were isolated, lacZ expression was silenced in 80% of the clones; however, lacZ expression was reactivated in all of the silenced clones by treatment with butyrate or trichostatin A. The two drugs also reactivated a silenced globin gene construct (rAAV͞HS2␣␤ AS3 ) in stably infected K562 cells. Trichostatin A is a specific inhibitor of histone deacetylase; therefore, we propose that hyperacetylation of histones after drug treatment changes the structure of chromatin on integrated viral sequences and relieves repression of transduced genes. The reactivation of silenced, transduced genes has implications for gene therapy. Efficient viral gene transfer followed by drug treatment to relieve suppression may provide a powerful combination for treatment of various genetic and infectious diseases.Retroviral and adeno-associated viral (AAV) vectors are two widely used viral systems for stably transferring genes into mammalian cells. Although the transfer of genes is generally efficient with these vectors, high-level, long-term expression in primary cells has been problematic. Palmer et al.(1) demonstrated efficient transfer of genes into primary skin fibroblasts with retroviral vectors, but expression was gradually suppressed over a period of 1 month, and a number of groups have observed inactivation of transferred ␤-globin genes after transduced bone marrow cells are transplanted into mice (Michel Sadelain, personal communication). These results are similar to the inhibition of retroviral expression observed by Jaenisch and colleagues after infection of preimplantation mouse embryos (2, 3). We recently demonstrated that retroviral long terminal repeat (LTR) sequences completely suppress ␤-globin gene expression in transgenic mice even in the presence of locus control region DNase I hypersensitive site 2 (HS2) sequences that normally direct high-level, position-independent expression (4). Severe inhibition is also observed when LTR HS3 ␤ constructs are tested in transgenic mice (James Ellis, personal communication). To localize sequences responsible for suppression, we inserted five separate subfragments of the retroviral LTR upstream of HS2␤ and tested these constructs for expression in mice. Surprisingly, four of the five fragments inhibited expression of the transgene (unpublished work); the only LTR sequence that did not inhibit expression was a fragment containing the retroviral enhancer and promoter.We recently tested AAV inverted terminal repeat (ITR) sequences in the assay...

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