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Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry
Nature Communications ( IF 16.6 ) Pub Date : 2020-10-16 , DOI: 10.1038/s41467-020-19047-7
Alexandra Stützer , Luisa M. Welp , Monika Raabe , Timo Sachsenberg , Christin Kappert , Alexander Wulf , Andy M. Lau , Stefan-Sebastian David , Aleksandar Chernev , Katharina Kramer , Argyris Politis , Oliver Kohlbacher , Wolfgang Fischle , Henning Urlaub

Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.



中文翻译:

紫外诱导交联和质谱分析染色质中的蛋白质-DNA相互作用

蛋白质与DNA的相互作用是基因组功能和稳定性的关键。蛋白质与DNA相互作用的界面和位点的鉴定和作图对于理解DNA依赖性过程至关重要。在这里,我们介绍了一个工作流程,该流程允许在通过紫外线(UV)交联后,与重组和天然染色质中的DNA直接接触的蛋白质进行质谱(MS)鉴定。我们的方法能够确定氨基酸水平的接触界面。以染色质相关蛋白SCML2为例,我们证明了我们的技术可以区分处于不同状态的核小体结合界面。通过分离核的UV交联,我们确定了多种因素的交联位点,包括染色质修饰酶,证明我们的工作流程不限于重组材料。由于我们的方法可以在一个实验中区分蛋白质与RNA和DNA的相互作用,因此我们预计将来可能会获得对染色质及其调控的见解。

更新日期:2020-10-17
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