Transactivation of the Xenopus rRNA gene promoter by its enhancer

Abstract

A key question concerning the mechanism of transcriptional activation by enhancers is about the role of the DNA that connects the enhancer to the promoter. The linking DNA will be important if a regulatory protein(s) binds to the enhancer and then tracks or slides along the DNA to the promoter, or if, on binding, the protein(s) alters the topological state of the DNA. By contrast, if the linking DNA loops out to allow the formation of a promoter-enhancer complex, or if the enhancer increases the local concentration of a transcription factor, co-linearity of the promoter and the enhancer will not be strictly required. In Xenopus laevis, the transcription of the ribosomal RNA genes is stimulated by an enhancer composed of repetitive sequences in the intergenic spacer regions. These repetitive elements contain 60 or 81 base pairs, and their activity is relatively independent of their position and orientation. When the enhancer and promoter sequences are each located on separate DNA molecules, however, the enhancer is no longer able to augment transcription. We have now tested whether or not this apparent requirement for having the enhancer and promoter in cis can be overcome by keeping them in close proximity while locating them separately on different molecules. This was achieved by generating multiply intertwined, dimeric-catenanes in which the enhancer and promoter were located in trans on different rings. By injecting these catenanes into frog oocytes and measuring the activity of the enhancers in a series of competition assays, we were able to demonstrate that such enhancers can augment transcription in vivo.