Hamperl Stephan, Brown Christopher R, Perez-Fernandez Jorge, Huber Katharina, Wittner Manuel, Babl Virginia, Stöckl Ulrike, Boeger Hinrich, Tschochner Herbert, Milkereit Philipp, Griesenbeck Joachim
Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Universität Regensburg, Regensburg, Germany.
Methods Mol Biol. 2014;1094:329-41. doi: 10.1007/978-1-62703-706-8_26.
Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.
目前大多数用于体内染色质分析的方法都依赖于对假定染色质成分或其翻译后修饰状态的先验知识。分离特定的天然染色体区域为获得对染色质的大致无偏分子描述提供了一种有吸引力的替代方法。在这里,我们描述了一种将染色体上的位点特异性重组与高效串联亲和纯化方案相结合的策略,以从酿酒酵母中分离单拷贝基因座。该方法能够有力地富集靶向染色质结构域,使其适用于成分、结构和生化分析。这项技术似乎适用于获得酵母中几乎任何基因组位点处染色质组成和特定翻译后组蛋白修饰状态的详细描述。