In vitro analysis of transcriptional repression of the mouse mammary tumorvirus promoter

Transcription of the proviral DNA of mouse mammary tumor virus (MMTV) is in duced by several classes of hormone-activated steroid receptor proteins. Ba sal promoter activity in the absence of receptor-mediated activation is sel ectively repressed by a distal negative regulatory element (dNRE) centered approximately 400 bp upstream of the transcription initiation site. An in v itro transcription system based on synthetic T-free cassette templates was developed to assess MMTV promoter activity, and dNRE-mediated repression wa s partially reconstituted with this system. Repression was observed with te mplates in which the dNRE was present in several sequence contexts. The act ivity of transcription preinitiation complexes formed in vitro in the prese nce of the dNRE could not be distinguished from that of complexes formed in its absence as assessed by the kinetics of transcript accumulation after a ddition of nucleoside triphosphates to preformed preinitiation complexes. d NRE-mediated repression in vitro appeared to be the result of decreased eff iciency of assembly of functional transcription complexes on the MMTV promo ter. However, repression could not be explained by inhibition of assembly o f TATA-binding protein or transcription factor HE into transcription comple xes, as neither protein decreased the extent of repression when supplied in excess as a purified recombinant protein. (C) 2001 Academic Press.