LOSS OF TRANSCRIPTIONAL ACTIVITY OF A TRANSGENE IS ACCOMPANIED BY DNAMETHYLATION AND HISTONE DEACETYLATION AND IS PREVENTED BY INSULATORS

The constitutive DNase I hypersensitive site at the 5' end of the chic ken beta-globin locus marks the boundary of the active chromatin domai n in erythroid cells. The DNA sequence containing this site has the pr operties of an insulator, as shown by its ability in stable transforma tion experiments to block enhancer-promoter interaction when it lies b etween the two, but not when it lies outside, and to protect against p osition effects in Drosophila. We now show that the chicken insulator can protect a stably integrated gene, which is otherwise subject to gr eat variability of expression, from chromatin-mediated repression in c ell culture. When the integrated reporter gene is surrounded by insula tor elements, stably transformed cell lines display consistent enhance r-dependent expression levels, in accord with the strength of the enha ncer. In the absence of insulators, long-term nonselective propagation of cells carrying the integrated reporter gene results in gradual ext inction of the reporter's expression, with expression patterns from ta ndemly repeated inserted genes suggesting that the extinction of adjac ent genes is coupled. We show that the uninsulated reporter genes, in addition to becoming transcriptionally inactive, lose several epigenet ic hallmarks of active chromatin, including nuclease accessibility, DN A hypomethylation, and histone hyperacetylation during time in culture . Treatment with inhibitors of histone deacetylase or DNA methylation reverses the extinction of the uninsulated genes. Extinction is comple tely prevented by flanking the reporter construct with insulators. Fur thermore, in contrast to the uninsulated reporter genes, chromatin ove r the insulated genes retains nuclease accessibility and histone hyper acetylation. However, there is no clear correlation between the presen ce of the insulators and the level of DNA methylation. This leads us t o propose a model for the insulator's ability to protect against extin ction in the transformed cell lines and to function as a chromatin bou ndary for the chicken beta-globin locus in normal erythroid cells.