In vivo binding of proteins to stably integrated MMTV DNA in murine cell lines: occupancy of NFI and OTF1 binding sites in the absence and presence of glucocorticoids.

Activation of expression at the mouse mammary tumor virus (MMTV) promoter is thought to be controlled by nucleosome positioning. On stably integrated MMTV DNA, the long terminal repeat (LTR) region is organized in a phased array of nucleosomes which allegedly occludes transcription factors such as NFI from binding. NFI only binds to the promoter region when the ordered nucleosome structure is apparently disrupted by activated steroid hormone receptors in hormone induced transcription. In certain cell lines, binding sites for the transcription factors NFI and OTF1 are however required for hormone-independent expression of MMTV. We have used stably transfected mouse NIH3T3 and GR cells that exhibit detectable MMTV expression in the absence of hormone for in vivo determination of proteins binding to the MMTV promoter. Here, we present in vivo dimethyl sulfate footprinting data that show that the NFI and OTF binding sites are permanently occupied in vivo in these cells. The contacting guanine residues identified in vivo were demonstrated in in vitro methylation interference assays to correspond to binding by NFI and OTF1. These results demonstrate a novel feature of transcription factor occupancy at the MMTV LTR promoter.