Binding of NF1 to the MMTV promoter in nucleosomes: influence of rotational phasing, translational positioning and histone H1

To analyse the role of rotational orientation and translational positioning of nucleosomal DNA on transcription factor binding we have generated a series of mutant MMTV promoters containing insertions of various lengths between the hormone-responsive region and the binding site for NF1. These various MMTV promoter fragments were assembled in mononucleosomes and used for structural studies and binding experiments, We show that the insertions change the rotational phase and translational positioning of the NF1 site as predicted if the sequences upstream of the insertion site were the main determinants of nucleosome phasing, In band shift experiments with recombinant NF1 we cannot detect binding of the protein to NF1 sites included within the limits of a nucleosome, independent of their rotational orientation, Moving the NF1 site closer to the nucleosome border also did not permit NF1 binding, This behaviour probably reflects the way NF1 binds DNA, namely it almost completely surrounds the circumference of the double helix establishing a large number of contacts with the bases and the backbone, In contrast to the wild-type and short insertion mutants, NF1 bound readily to nucleosomes containing 30 or 50 bp insertions which placed the NF1 site at the nucleosome edge or within linker DNA, NF1 binding to the linker DNA was unaffected by incorporation of histone H1 into the nucleosome particle, These findings are discussed in relation to chromatin remodelling initiated by steroid hormones during induction of the MMTV promoter.