Nucleic Acids Research: VOLUME 39 ISSUE 15 2011

Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltkfibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. INTRODUCTION Transcription from the proviral DNA of mouse mammary tumor virus (MMTV) is controlled by steroid hormones (1,2) through their binding to receptor proteins and the interaction of hormone-receptor complexes with regulatory DNA sequences, thereby causing an increase of the number of active RNA polymerase II molecules on the MMTV promoter (3). The hormone-responsive sequences are located in the long terminal repeat (LTR) of the provirus (4,5,6,7), in a region of approximately 200 bp upstream of the transcription start site (8,9,10). While steroids like progestins and androgens have recently been shown to also affect MMTV transcription in mammary cells (11,12,13,14,15), it is the response to glucocorticoids in cultured fibroblasts that has been studied in greatest detail (16,17,18,19). In vitro binding of purified glucocorticoid receptor to MMTV DNA has been demonstrated, showing several binding sites within the glucocorticoid regulatory region (20,21,22). Several reasons make MMTV a particularly suitable model system for studying transcription regulation by glucocorticoid hormones. High stimulation factors (200-fold and more; ref. 19) are observed after addition of dexamethasone, a synthetic glucocorticoid, to cells containing the natural MMTV promoter plus the regulatory region. Stimulation occurs over a low basal promoter activity which is independent of the presence of hormone or of hormone-responsive sequences (8,9). These sequences are located relatively close to the promoter and contain several regulatory elements which act in a cooperative fashion to achieve an optimal level of transcription (18). In a previous study using linker-insertion mutants (18) we have identified four distinct control elements for glucocorticoid regulation (Fig. 1A). Two of them (a distal one, between -18I and -172, and a proximal one, 3065 Nucleic Acids Research Volume 17 Number 8 1989