Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.
Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland.
Nat Protoc. 2017 Sep;12(9):1951-1961. doi: 10.1038/nprot.2017.072. Epub 2017 Aug 24.
Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.
蛋白质 ADP-核糖基化是一种结构异构的翻译后修饰（PTM），它影响修饰蛋白的理化性质和生物学特性。染色质的 ADP-核糖基化改变了其结构特性，从而调节重要的核功能。由于缺乏适用于染色质免疫沉淀（ChIP）的抗体，迄今为止，还无法对与 DNA 相关的蛋白 ADP-核糖基化进行全面分析。为了分析染色质 ADP-核糖基化，我们最近开发了一种新的 ADP-核糖基特异性染色质亲和纯化（ADPr-ChAP）方法，该方法使用最近鉴定的 ADP-核糖结合结构域 RNF146 WWE 和 Af1521。在本方案中，我们将描述如何使用这种强大且通用的方法进行全基因组和特定基因座的染色质 ADP-核糖基化定位。ADPr-ChAP 可实现 ADP-核糖基化与其他染色质修饰的生物信息学比较，有助于了解 ADP-核糖基化如何调节对生物学重要的细胞过程。ADPr-ChAP 大约需要 1 周的时间，并且需要具备分子生物学和生物化学方面的标准技能。尽管这里没有详细介绍，但该技术还可以与常规 ChIP 或 DNA 分析相结合，以定义与 ADP-核糖基化染色质分数特异性相关的组蛋白标记，并剖析染色质 ADP-核糖基化的分子机制和功能作用。
