A mouse mammary tumor virus promoter element is composed of sequences near the transcription initiation site

Abstract

Transcription from the mouse mammary tumor virus (MMTV) promoter is regulated through the complex interaction of regulatory and promoter elements. Analysis of the promoter using a transient transfection assay has identified several regions that are sensitive to mutation. This approach has certain limitations which can be overcome through the use of an in vitro transcription assay. In an attempt to identify and characterize proteins required for regulating transcription, an in vitro system derived from bovine liver nuclear extract was developed which is capable of transcribing MMTV. The sensitivity of the assay to the mycotoxin α-amanitian confirms that RNA polymerase II is responsible for transcription. Mutations which affect the activity of the promoter in vivo show a comparable decrease in transcription when tested in vitro. One of the mutations, which changes positions -2 to +6, results in a significant drop in activity both in vivo and in vitro. Using electrophoresis mobility shift and DNase I footprinting experiments, a protein is shown to bind to sequences between -12 and +13. This protein has been termed initiation site binding protein (ISBP). Efficient transcription from the MMTV promoter requires binding of ISBP as demonstrated by a competition assay in which an oligonucleotide containing sequences from -16 to +17 was able to inhibit promoter activity in vitro. To further define the bases needed for ISBP binding and activity, 58 plasmids with mutations between -4 and +10 were constructed. These mutant promoters were tested in a mobility shift assay and used as templates in the in vitro transcription reaction. The bases that were the most sensitive to mutation were between +5 and +10. This sequence of 5'-CAGTCC-3' defines the core region for ISBP binding and transcriptional activity. The SV40 late and dihydrofolate reductase (DHFR) promoters have functionally significant sequences at their start sites that are homologous to the core region defined for ISBP binding. To determine if the protein interacting with the SV40 and DHFR promoters was the same as ISBP, a series of binding competition experiments was performed. The ability of the SV40 and DHFR sequences to affect in vitro transcription from the MMTV promoter was also tested. The results of these experiments suggest that ISBP is a distinct protein from the one(s) that interacts with the SV40 and DHFR promoters.