The DNA of MMTV LTR is positioned on a phased array of nucleosomes (A-F). Hormone activation of the MMTV promoter leads to modulation of nucleosome B structure in vivo which leads to a region of DNA hypersensitive to nucleolytic reagents. High-resolution analysis of nucleosome position using PCR amplification indicates that the nucleosomes are positioned at base pair resolution. Hyper-sensitivity results from alteration in DNA structure across the B nucleosome and into the A-B linker region. This transition in structure is now known to be directly involved in transcription preinitiation complex recruitment (see project new-1 LMV). Four new assays were developed for positioning of nucleosomes in vivo and in vitro. The results from these new techniques indicate that the path of DNA on the nucleosomal surface is quite specific and reproducible, and has major ramifications for gene activation and repression. Hormone induction leads to H1 depletion from the A-B region in stable chromatin, whereas the core histone complement of this region is unchanged. The F nucleosome has also been positioned at high resolution; a gap in the nucleosome array occurs between F and the end of the MMTV LTR that apparently permits the binding of tissue-specific transcription factors (see project Z01CP05660-03 LMV) without the major structural transition required at the A-B region. These results indicate that a chromatin template containing specifically positioned nucleosomes is an active participant in transcriptional activation, and that modulation of this template structure is one feature of steroid hormone action.
