Orlando V, Strutt H, Paro R
ZMBH, Center for Molecular Biology, University of Heidelberg, Germany.
Methods. 1997 Feb;11(2):205-14. doi: 10.1006/meth.1996.0407.
Recent advances leave no doubt that higher order chromatin structures play a fundamental role in many developmentally important mechanisms of gene regulation. In particular analyses in genetic model systems like yeast and Drosophila uncovered novel proteins that are involved in the regulation of chromatin structures. Many of these proteins do not bind directly to DNA but interact in large multimeric complexes. To identify the DNA elements regulated by these multiprotein complexes, alternative approaches to the standard methods of DNA-protein analysis had to be devised. Here we present a method that preserves the architecture of the higher order chromatin structures by cross-linking cells in vivo with formaldehyde. An immunoprecipitation strategy is then used to identify the DNA targets of chromosomal proteins of interest. This method can be applied to study the distribution of proteins at high resolution over extended chromosomal regions.
近期的研究进展无疑表明，高阶染色质结构在许多对发育至关重要的基因调控机制中发挥着根本性作用。特别是在酵母和果蝇等遗传模型系统中的分析，发现了参与染色质结构调控的新型蛋白质。这些蛋白质中的许多并不直接与DNA结合，而是在大型多聚体复合物中相互作用。为了鉴定由这些多蛋白复合物调控的DNA元件，必须设计出不同于标准DNA-蛋白质分析方法的替代方法。在此，我们提出一种通过在体内用甲醛交联细胞来保留高阶染色质结构架构的方法。然后采用免疫沉淀策略来鉴定感兴趣的染色体蛋白质的DNA靶点。该方法可用于在扩展的染色体区域上高分辨率地研究蛋白质的分布。
