We have studied enhancer function in transient and stable expression assays in mammalian cells by using systems that distinguish expressing from nonexpressing cells. When expression is studied in this way, enhancers are found to increase the probability of a construct being active but not the level of expression per template. In stably integrated constructs, large differences in expression level are observed but these are not related to the presence of an enhancer. Together with earlier studies, these results suggest that enhancers act to affect a binary (on/off) switch in transcriptional activity. Although this idea challenges the widely accepted model of enhancer activity, it is consistent with much, if not all, experimental evidence on this subject. We hypothesize that enhancers act to increase the probability of forming a stably active template. When randomly integrated into the genome, enhancers may affect a metastable state of repression/activity, permitting expression in regions that would not permit activity of an isolated promoter.
