Epigenetics and Cell Fate Centre, UMR7216 CNRS, Université de Paris, Paris, France.
Novo Nordisk Foundation Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Nat Protoc. 2021 Sep;16(9):4446-4493. doi: 10.1038/s41596-021-00585-3. Epub 2021 Aug 6.
Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.
阐明基因组复制后染色质维持的机制对于理解基因表达程序和细胞身份如何在细胞分裂中得以保留至关重要。在这里，我们描述了两种最近开发的技术，复制后染色质占有率（ChOR-seq）和复制后姐妹染色单体（SCAR-seq），它们在 5 天的实验工作中在哺乳动物细胞中对新复制 DNA 上的染色质占有率进行了分析。这两种技术都采用了一种共同的策略，包括新合成 DNA 的脉冲标记和染色质免疫沉淀（ChIP），然后进行纯化和高通量测序。ChOR-seq 定量分析了复制后组蛋白修饰和染色质相关蛋白的丰度，而 SCAR-seq 则区分了新生姐妹染色单体之间的染色质占有率。这两种互补技术共同揭示了控制复制过程中修饰组蛋白遗传的关键机制，并揭示了整个细胞周期中组蛋白修饰的特定基因座动态。在这里，我们提供了这些方法的实验方案和生物信息学流程。
