Nucleic Acids Research, Vol 25, Issue 18 3733-3742, Copyright © 1997 by Oxford University Press
K Eisfeld, R Candau, M Truss and M Beato
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.
ARTICLES
Binding of NF1 to the MMTV promoter in nucleosomes: influence of rotational phasing, translational positioning and histone H1
Institut fur Molekularbiologie und Tumorforschung, Philipps Universitat, E.-Mannkopff-Strasse 2, 35037 Marburg, Germany.
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