Spatial Compartmentalization at the Nuclear Periphery Characterized by Genome-wide Mapping

How gene positioning to the nuclear periphery regulates transcription remains largely unclear. We have previously observed the differential compartmentalization of transcription factors and histone modifications at the nuclear periphery in mouse C2C12 myoblasts. Here, we have integrated high throughput DNA sequencing into the DNA adenine methyltransferase identification (DamID) assay, and have identified ~15, 000 sequencing-based Lamina-Associated Domains (sLADs) in mouse 3T3 fibroblasts and C2C12 myoblasts. These genomic regions range from a few kb to over 1 Mb and cover ~30% of the genome, and are spatially proximal to the nuclear lamina (NL). Active histone modifications such as H3K4me2, H3K9Ac, H3K36me3 and H3K79me2 are all localized away from the nuclear periphery microscopically, and distributed predominantly out of sLADs genome-wide. Therefore, the spatial compartmentalization of active histone modifications likely characterizes a major portion of chromatin at the nuclear periphery in mammalian cells. Genomic regions around transcription start sites of expressed sLAD genes display reduced associations with the NL and possess active histone modifications; in contrast, gene bodies of expressed sLAD genes possess very low levels of active histone modifications. Our genome-wide analyses of NL-associated chromatin have enabled functional and mechanistic dissections of gene positioning on transcription regulation. Overall design: generate DamID maps of genome-NL interaction for mouse 3T3 fibroblasts and C2C12 myoblasts with two control samples (*genomic DNA samples).

基因定位于核外周后如何调控转录，目前仍未完全阐明。我们此前已在小鼠C2C12成肌细胞中观察到，转录因子与组蛋白修饰在核外周存在差异化的区室化分布。本研究将高通量DNA测序整合至DNA腺嘌呤甲基转移酶鉴定法（DNA adenine methyltransferase identification, DamID）实验体系中，在小鼠3T3成纤维细胞与C2C12成肌细胞内鉴定得到约15000个测序版核纤层关联结构域（sequencing-based Lamina-Associated Domains, sLADs）。此类基因组区域的跨度从数kb至1 Mb以上，覆盖约30%的小鼠基因组，且在空间上紧邻核纤层（nuclear lamina, NL）。显微镜观察显示，H3K4me2、H3K9Ac、H3K36me3及H3K79me2等活性组蛋白修饰均不富集于核外周区域，且在全基因组范围内主要分布于sLADs之外。由此可见，活性组蛋白修饰的空间区室化分布，大概率是哺乳动物细胞核外周染色质的重要特征之一。表达型sLAD基因的转录起始位点周边基因组区域，与NL的结合水平显著降低，且富集活性组蛋白修饰；与之相反，该类基因的基因本体区域仅携带极低水平的活性组蛋白修饰。本研究通过对NL结合染色质的全基因组分析，实现了基因定位对转录调控作用的功能与机制解析。实验整体设计：针对小鼠3T3成纤维细胞与C2C12成肌细胞，构建基因组-核纤层互作的DamID图谱，同时设置两份对照样本（*基因组DNA样本）。