Agudelo Garcia Paula A, Gardner Miranda, Freitas Michael A, Parthun Mark R
Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, USA.
Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2019;1983:17-27. doi: 10.1007/978-1-4939-9434-2_2.
Replication-coupled chromatin assembly is a very dynamic process that involves not only the replication fork machinery but also chromatin-related factors such as histones, histone chaperones, histone-modifying enzymes, and chromatin remodelers which ensure not only that the genetic information is properly replicated but also that the epigenetic code is reestablished in the daughter cell. Of the histone modifications associated with chromatin assembly, acetylation is the most abundant. Determining how newly synthesized histones get acetylated and what factors affect this modification is vital to understanding how cells manage to properly duplicate the epigenome. Here we describe a combination of the iPOND, quantitative mass spectrometry, and SILAC methodologies to study the protein composition of newly assembled chromatin and the modification state of the associated histones.
复制偶联染色质组装是一个非常动态的过程，不仅涉及复制叉机制，还涉及染色质相关因子，如组蛋白、组蛋白伴侣、组蛋白修饰酶和染色质重塑因子，这些因子不仅确保遗传信息得到正确复制，还确保表观遗传密码在子细胞中得以重新建立。在与染色质组装相关的组蛋白修饰中，乙酰化最为丰富。确定新合成的组蛋白如何被乙酰化以及哪些因素影响这种修饰，对于理解细胞如何成功地正确复制表观基因组至关重要。在这里，我们描述了一种结合iPOND、定量质谱和SILAC方法来研究新组装染色质的蛋白质组成以及相关组蛋白的修饰状态。
