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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Mutations in mouse and human patched1 (ptc1) genes are associated with birth defects and cancer. Ptc1 is a receptor for Hedgehog (Hh) signaling proteins. Hh proteins activate transcription of target genes, including ptc1, and Ptc1 represses those genes, both by regulating the activity of Gli transcription factors. We have established mammalian cell lines with reduced Ptc1 function and a lacZ reporter to investigate Hh signal transduction. Embryonic fibroblasts were derived from mice, heterozygous or homozygous for a ptc1 mutation that inserts lacZ under the control of the ptc1 promoter (ptc1-lacZ). In heterozygous ptc1 cells, ptc1-lacZ was expressed at low levels but could be induced by Sonic Hedgehog (Shh) and Gli-1. Homozygous ptc1 cells expressed high levels of ptc1-lacZ without Hh stimulation. ptc1-lacZ expression was dependent on cell density in ptc1 homozygotes and Hh-stimulated heterozygotes but was independent of density when Gli1 was used to activate ptc1-lacZ. A wild-type ptc1 transgene introduced into homozygous ptc1 cells greatly reduced ptc1-lacZ expression. Expression of either half of Ptc1 alone resulted in improper maturation of the protein and a failure to complement the ptc1 ؊/؊ cells. When co-expressed, both Ptc1 halves matured and had an activity similar to that of the intact protein. Three missense PTCH1 mutations exhibited significant functions in homozygous ptc1 cells. The missense mutants retained activity when expressed at about 10-fold lower levels and appeared as stable as wild-type Ptc1. These studies suggest that some tumors and disease phenotypes may arise from small reductions in PTCH1 activity.

The membrane protein Patched (Ptc) is a critical regulator of Hedgehog signaling. Ptc is among a family of proteins that contain a sterol sensor motif. The function of this domain is poorly understood, but some proteins that contain sterol sensors are involved in cholesterol homeostasis. In the SREBP cleavage-activating protein (SCAP), sterols inhibit the protein's activity through this domain. Mutations in two highly conserved residues in the SCAP sterol sensor have been identified that confer resistance to sterol regulation. We introduced the analogous mutations in the sterol sensor motif of fly Ptc and mouse Ptc1 and examined their effect on protein activity. In contrast to SCAP, the sterol sensor mutations had different affects on Drosophila Ptc; Ptc Y442C retained function, while Ptc D584N conferred dominant negative activity. In the wing imaginal disc, Ptc D584N overexpression induced Hedgehog targets by stabilizing Cubitus interruptus and inducing decapentaplegic. However, Ptc D584N did not induce collier, a gene that requires high levels of Hedgehog signaling. In mouse Ptc1, the Y438C and D585N mutations did not stimulate signaling in Shh-responsive cell lines but did complement murine ptc1(-/-) cells. The results suggest that mutations in sterol sensor motifs alter function differently between sterol sensor family members.