Spangenberg C, Eisfeld K, Stünkel W, Luger K, Flaus A, Richmond T J, Truss M, Beato M
Institut für Molekularbiologie und Tumorforshung, Philipps-Universität, Marburg, Germany.
J Mol Biol. 1998 May 15;278(4):725-39. doi: 10.1006/jmbi.1998.1718.
Modulation of eukaryotic gene expression is influenced by the organization of regulatory DNA-elements in chromatin. The mouse mammary tumor virus (MMTV) promoter exhibits regularly positioned nucleosomes that reduce the accessibility of the binding sites for sequence-specific transcription factors, in particular nuclear factor (NF1). Hormonal induction of the MMTV promoter is accompanied by remodeling of the nucleosomal structure, but the biochemical nature of these structural changes is unknown. Using recombinant histones, we have now assembled the MMTV promoter in particles containing either an octamer of the histones H3, H4, H2A and H2B or a tetramer of histones H3 and H4, and have compared the two particles in terms of structure, positioning, and exclusion of transcription factors. Using site-directed hydroxy radicals to map histone locations, two main nucleosome positions are found with dyads at position -107 and at -127. The same two main positions are found for particles containing only the H3/H4 tetramer, showing that the absence of H2A/H2B dimers does not alter positioning. The rotational orientation of the DNA double helix in both types of particles is essentially identical. However, the ends of the nucleosomal DNA as well as its central region are more accessible to cleavage reagents in the tetramer particle than in the octamer particle. In agreement with these structural features, the transcription factors NF1 and OTF1 were able to bind to their cognate sites on the tetramer particle, while they could not gain access to the same sites on the surface of the octamer particle. The DNase I digestion pattern of octamers treated with partially purified SWI/SNF complex from HeLa cells in the presence of ATP is indistinguishable from that of tetramer particles, suggesting that the SWI/SNF complex promotes ATP-dependent remodeling of the octamer particle but not of tetramer particles. These results are compatible with a hormone-induced removal of histone H2A/H2B during MMTV induction.
真核基因表达的调控受染色质中调控DNA元件组织方式的影响。小鼠乳腺肿瘤病毒（MMTV）启动子具有规则定位的核小体，这些核小体会降低序列特异性转录因子（特别是核因子（NF1））结合位点的可及性。MMTV启动子的激素诱导伴随着核小体结构的重塑，但这些结构变化的生化本质尚不清楚。我们现在使用重组组蛋白，在含有组蛋白H3、H4、H2A和H2B八聚体或组蛋白H3和H4四聚体的颗粒中组装了MMTV启动子，并比较了这两种颗粒在结构、定位和转录因子排斥方面的情况。使用定点羟基自由基来绘制组蛋白位置，发现了两个主要的核小体位置，其二联体分别位于-107和-127位置。对于仅含有H3/H4四聚体的颗粒也发现了相同的两个主要位置，这表明缺少H2A/H2B二聚体不会改变定位。两种颗粒中DNA双螺旋的旋转方向基本相同。然而，与八聚体颗粒相比，四聚体颗粒中核小体DNA的末端及其中心区域对切割试剂更易接近。与这些结构特征一致，转录因子NF1和OTF1能够结合到四聚体颗粒上它们的同源位点，而它们无法接近八聚体颗粒表面的相同位点。在ATP存在的情况下，用来自HeLa细胞的部分纯化的SWI/SNF复合物处理的八聚体的DNase I消化模式与四聚体颗粒的消化模式无法区分，这表明SWI/SNF复合物促进八聚体颗粒的ATP依赖性重塑，但不促进四聚体颗粒的重塑。这些结果与MMTV诱导过程中激素诱导的组蛋白H2A/H2B去除相一致。
