Assembly of MMTV promoter minichromosomes with positioned nucleosomes precludes NF1 access but not restriction enzyme cleavage.
To generate long arrays of nucleosomes within a topologically defined chromatin domain we have assembled minichromosomes on negatively supercoiled plasmid DNA with extracts from Drosophila preblastoderm embryos. These minichromosomes are dynamic substrates for energy-dependent nucleosome remodeling machines that facilitate the binding of various transcription factors but do not exhibit nucleosome positioning. In contrast, if such minichromosomes include the mouse mammary tumour virus (MMTV) promoter we find it wrapped around a nucleosome with similar translational and rotational position as in vivo . This structure precluded binding of NF1 to its cognate site at -75/-65 at salt concentrations between 60 and 120 mM, even in the presence of ATP, which rendered the NF1 site accessible to the restriction enzyme Hin fI. However, insertion of 30 bp just upstream of the NF1 site, which moves the site to the linker DNA, allowed ATP-dependent binding of NF1 to a fraction of the minichromosomes, even in the presence ofstoichiometric amounts of histone H1. The minichromosomes assembled in the Drosophila embryo extract reproduce important features of the native MMTV promoter chromatin and reveal differences in the ability of transcription factors and restriction enzymes to access their binding sites in positioned nucleosomes.
IMT, Institut für Molekularbiologie und Tumorforschung, Marburg, Emil-Mannkopff-Strasse 2, D-35033 Marburg, Germany and EMBL, Meyerhofstrasse 1, D-649117 Heidelberg, Germany.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004330:Drosophila / Q000196:embryology / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D016254:Mutagenesis, Insertional; D051839:NFI Transcription Factors; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D002843:Chromatin; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006657:Histones; D051839:NFI Transcription Factors; D009707:Nucleosomes; D011994:Recombinant Proteins; D014157:Transcription Factors; C080333:transcription factor nuclear factor 1; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases