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 NFkappaB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFkappaB 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 & Dev. 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 Sp1 and NFkappaB 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 NFkappaB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFkappaB. 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 NFkappaB binding sites.
Highlights
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 NFkB binding sites
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
We extend these earlier studies by demonstrating that the process is ATPdependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation
Summary
Plasmid Constructs and DNA Labeling—The HIV-1 SphI/SphI fragment of pHXB2 [28] was inserted into pNEB193 (lacking a AvaI polylinker site). A 10-ml reaction mixture consisted of 4 ml of oocyte extract, 15–25 ng of labeled DNA (the optimal amount was experimentally determined for each DNA preparation), 10 ng of creatine phosphokinase, and 1 ml of 10 3 chromatin assembly buffer (400 mM creatine phosphate, 20 mM ATP, 10 mM MgCl2). Final concentrations of the factors in the assembly mixtures were as follows: 10 nM Sp1, 50 nM NFkB (homo- or heterodimer), 50 nM AP1 (c-Jun homodimer), 50 nM TBP, and 100 nM Tat. In other experiments Sp1 (20 nM) and NFkB (100 nM) were added after chromatin assembly, and the reaction was allowed to proceed for another 30 min. ATP was removed from reaction mixtures that contained assembled minichromosomes according to Tsukiyama et al [36]; mixtures were either treated with apyrase (final concentration 0.01 unit/ml) for 15 min at 27 °C or filtered through spin columns containing Bio-Gel A-1.5m (Bio-Rad). To analyze the nucleosomal ladders of minichromosomes, CaCl2 (final concentration 3 mM) and micrococcal nuclease (Worthington, final concentration 0.15 unit/ml)
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