Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their normal chromosomal context. They stimulate transcription in an orientation-independent manner when assayed on circular plasmids. We describe a transient transformation system to evaluate the orientation preference of transcriptional enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to block bidirectional action of an enhancer on circular plasmids. In this system, as in the chromosome, blocking of enhancer activity requires wild-type levels of the su(Hw) protein. We evaluated the orientation preference for the relatively large (4.4 kb) Adh larval enhancer from Drosophila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revealed by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promoter was weakened by removing binding sites for critical transcription factors, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The orientation preference, as much as 100-fold, is a property of the enhancer itself because it is displayed by gene constructions introduced into the chromosome regardless of the presence of the insulator in a distal location. These findings are most easily reconciled with a facilitated tracking mechanism for enhancer function in a native chromosomal environment
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