Krietenstein Nils, Wippo Christian J, Lieleg Corinna, Korber Philipp
Adolf-Butenandt-Institut, Molecular Biology Unit, University of Munich, Munich, Germany.
Methods Enzymol. 2012;513:205-32. doi: 10.1016/B978-0-12-391938-0.00009-4.
Recent genome-wide mapping of nucleosome positions revealed that well-positioned nucleosomes are pervasive across eukaryotic genomes, especially in important regulatory regions such as promoters or origins of replication. As nucleosomes impede access to DNA, their positioning is a primary mode of genome regulation. In vivo studies, especially in yeast, shed some light on factors involved in nucleosome positioning, but there is an urgent need for a complementary biochemical approach in order to confirm their direct roles, identify missing factors, and study their mechanisms. Here we describe a method that allows the genome-wide in vitro reconstitution of nucleosomes with very in vivo-like positions by a combination of salt gradient dialysis reconstitution, yeast whole cell extracts, and ATP. This system provides a starting point and positive control for the biochemical dissection of nucleosome positioning mechanisms.
近期对核小体位置进行的全基因组图谱绘制显示，定位良好的核小体普遍存在于真核生物基因组中，尤其是在重要的调控区域，如启动子或复制起点。由于核小体阻碍了对DNA的访问，它们的定位是基因组调控的主要方式。体内研究，特别是在酵母中的研究，揭示了一些参与核小体定位的因素，但迫切需要一种互补的生化方法来确认它们的直接作用、识别缺失的因素并研究其机制。在这里，我们描述了一种方法，该方法通过盐梯度透析重构、酵母全细胞提取物和ATP的组合，能够在全基因组范围内体外重构出具有非常类似体内位置的核小体。该系统为核小体定位机制的生化剖析提供了一个起点和阳性对照。
