Venditti P, Di Croce L, Kauer M, Blank T, Becker P B, Beato M
IMT, Institut für Molekularbiologie und Tumorforschung, Marburg, Emil-Mannkopff-Strasse 2, D-35033 Marburg, Germany and EMBL, Meyerhofstrasse 1, D-649117 Heidelberg, Germany.
Nucleic Acids Res. 1998 Aug 15;26(16):3657-66. doi: 10.1093/nar/26.16.3657.
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.
为了在拓扑结构确定的染色质结构域内生成长链核小体，我们利用果蝇胚盘前胚胎提取物在负超螺旋质粒DNA上组装了微型染色体。这些微型染色体是能量依赖型核小体重塑机器的动态底物，有助于各种转录因子的结合，但不表现出核小体定位。相比之下，如果这些微型染色体包含小鼠乳腺肿瘤病毒（MMTV）启动子，我们会发现它与体内情况类似，以相似的平移和旋转位置包裹在一个核小体周围。这种结构阻止了NF1在60至120 mM盐浓度下结合其位于-75/-65的同源位点，即使存在ATP时也是如此，这使得NF1位点可被限制性内切酶Hin fI识别。然而，在NF1位点上游插入30 bp，将该位点移至连接区DNA，即使在存在化学计量的组蛋白H1时，也能使NF1以ATP依赖的方式结合一部分微型染色体。在果蝇胚胎提取物中组装的微型染色体重现了天然MMTV启动子染色质的重要特征，并揭示了转录因子和限制性内切酶在定位核小体中结合其位点能力的差异。
