RNA Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, 351-0198, Japan.
RNA Systems Biochemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, 351-0198, Japan.
Sci Rep. 2018 Jan 18;8(1):1143. doi: 10.1038/s41598-018-19494-9.
The nervous system of higher eukaryotes is composed of numerous types of neurons and glia that together orchestrate complex neuronal responses. However, this complex pool of cells typically poses analytical challenges in investigating gene expression profiles and their epigenetic basis for specific cell types. Here, we developed a novel method that enables cell type-specific analyses of epigenetic modifications using tandem chromatin immunoprecipitation sequencing (tChIP-Seq). FLAG-tagged histone H2B, a constitutive chromatin component, was first expressed in Camk2a-positive pyramidal cortical neurons and used to purify chromatin in a cell type-specific manner. Subsequent chromatin immunoprecipitation using antibodies against H3K4me3-a chromatin modification mainly associated with active promoters-allowed us to survey the histone modifications in Camk2a-positive neurons. Indeed, tChIP-Seq identified hundreds of H3K4me3 modifications in promoter regions located upstream of genes associated with neuronal functions and genes with unknown functions in cortical neurons. tChIP-Seq provides a versatile approach to investigating the epigenetic modifications of particular cell types in vivo.
高等真核生物的神经系统由众多类型的神经元和神经胶质细胞组成，它们共同协调复杂的神经元反应。然而，在研究特定细胞类型的基因表达谱及其表观遗传基础时，这个复杂的细胞群体通常会带来分析上的挑战。在这里，我们开发了一种新的方法，即串联染色质免疫沉淀测序（tChIP-Seq），可以对表观遗传修饰进行特定细胞类型的分析。FLAG 标记的组蛋白 H2B 是一种组成性染色质成分，首先在 Camk2a 阳性皮质锥体细胞中表达，以特定细胞类型的方式纯化染色质。随后使用针对 H3K4me3 的抗体进行染色质免疫沉淀 - H3K4me3 是一种主要与活跃启动子相关的染色质修饰 - 使我们能够检测 Camk2a 阳性神经元中的组蛋白修饰。事实上，tChIP-Seq 鉴定了皮质神经元中与神经元功能相关的基因和功能未知基因的上游启动子区域内数百个 H3K4me3 修饰。tChIP-Seq 为研究特定细胞类型的表观遗传修饰提供了一种通用的方法。
