Position effects are influenced by the orientation of a transgene with respect to flanking chromatin

We have inserted two expression cassettes at tagged reference chromosomal s ites by using recombinase-mediated cassette exchange in mammalian cells, Th e three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cass ettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive, Orientation-specific silencin g, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to swi tch in vivo the orientation of the transgenes from the permissive to the no npermissive orientation and vice versa, Switching from the permissive to th e nonpermissive orientation led to silencing, but switching from the nonper missive to the permissive orientation did not lead to reactivation of the t ransgene. Instead, transgene expression occurred dynamically by transcripti onal oscillations, with 10 to 20% of the cells expressing at any given time . This result suggested that the cassette had been imprinted (epigeneticall y tagged) while it was in the nonpermissive orientation. Methylation analys is revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost so me of its methylation. Sorting of the expressing and nonexpressing cell pop ulations provided evidence that the transcriptional oscillations of the epi genetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene, This suggests that transgen e methylation is more dynamic than was previously assumed.