In vitro chromatin assembly of the HIV-1 promoter: ATP-dependent polar repositioning of nucleosomes by Sp1 and NFκB

Piotr Widlak, Richard B. Gaynor, William T. Garrard

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Abstract

Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5'- long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFκB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFκB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes and Der. 10, 37-49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Spl and NFκB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFκB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFκB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFκB binding sites.

Original languageEnglish (US)
Pages (from-to)17654-17661
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number28
DOIs
StatePublished - Aug 5 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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