THE HORMONE-RESPONSIVE REGION OF MOUSE MAMMARY-TUMOR VIRUS POSITIONS A NUCLEOSOME AND PRECLUDES ACCESS OF NUCLEAR FACTOR-I TO THE PROMOTER

The mouse mammary tumor virus (MMTV) promoter is transcriptionally sil ent prior to hormonal induction, partly because its organization into phased nucleosomes precludes access of transcription factors to their cognate sites. A T47D-derived cell line carrying a single integrated c opy of the MMTV promoter exhibited a positioned nucleosome, which prev ented binding of nuclear factor I (NFI) [1]. To study the molecular me chanisms controlling promoter accessibility we have made use of a stro ng chimeric transactivator, NFI-VP16, composed of NFI linked to the tr ansactivation function of VP16. T47D cells transiently transfected wit h an MMTV-CAT reporter show little transcription even after cotransfec tion of an expression vector for NFI-VP16. However, a truncated MMTV p romoter, lacking the hormone regulatory region (HRR) was transactivate d by cotransfected NFI-VP16. The repressive effect of the HRR was not due to binding of a sequence-specific transcriptional repressor, and w as evident with the DEAE-Dextran transfection procedure but not with t he calcium phosphate technique. A similar behavior was observed in Sac charomyces cerevisiae carrying wild type or truncated MMTV-lacZ report ers and expressing NFI-VP16. Reconstitution experiments suggest that t he promoter lacking the HHR generates less stable nucleosomes, a fract ion of which contain a more accessible NFI site. Recombinant NFI binds to nucleosomes assembled on this truncated promoter but not to nucleo somes encompassing the HRR. These results are compatible with the noti on that transiently transfected MMTV promoters behave like their stabl y integrated counterparts, in that the HRR drives positioning of a nuc leosome and mediates transcriptional repression by preventing access o f NFI to its cognate site. (C) 1996 Elsevier Science Ltd.