Polarity of transcriptional enhancement revealed by an insulator element

Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their norma l chromosomal context. They stimulate transcription in an orientation-indep endent manner when assayed on circular plasmids. We describe a transient tr ansformation system to evaluate the orientation preference of transcription al enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to black bidirectional action of an enhancer on circular plasmids . In this system, as in the chromosome, blocking of enhancer activity requi res wild-type levels of the su(Hw) protein. We evaluated the orientation pr eference for the relatively large (4.4 kb) Adh larval enhancer from Drosoph ila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revea led by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promo ter was weakened by removing binding sites for critical transcription facto rs, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The or ientation preference, as much as 100-fold, is a property of the enhancer it self because it is displayed by gene constructions introduced into the chro mosome regardless of the presence of the insulator in a distal location. Th ese findings are most easily reconciled with a facilitated tracking mechani sm for enhancer function in a native chromosomal environment.