LaminB1 ChIP-seq of DP thymocytes from wildtype and Suv39h1 and Suv39h2 double knockout mice. LaminB1 ChIP-seq of DP thymocytes from wildtype and Suv39h1 and Suv39h2 double knockout mice

H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Within all eukaryotic nuclei, heterochromatin and euchromatin are spatially segregated, with euchromatin typically located in the nuclear interior and heterochromatin at the nuclear periphery. Here we investigate the disruption of lamina-association in the absence of H3K9me3-dependent heterochromatin by performing LaminB1 ChIP-seq in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition. Overall design: CD4+CD8+ double positive mouse thymocyte cells were analysed. ChIP and whole-cell extract (WCE) libraries from two replicates from Suv39h1+/y Suv39h2+/- (control) mice and two replicates from Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice were independently prepared and sequenced to confirm successful immunoprecipitation. For the bioinformatics analysis, the replicate FASTQ files were merged. LaminB1 peaks were then called separately for the knockout and control mice using WCE as the input control in each case.

依赖H3K9me3的异染色质（H3K9me3-dependent heterochromatin）对于富含重复序列的着丝粒周边区域的基因沉默至关重要，同时在抑制与细胞谱系不匹配的蛋白编码基因以维持细胞正常生理功能方面也发挥关键调控作用。在所有真核生物的细胞核中，异染色质与常染色质存在空间分布上的分隔：常染色质通常定位于细胞核内部，而异染色质则聚集于核膜边缘区域。本研究通过在同时缺失Suv39h1与Suv39h2的原代免疫细胞（Suv39DKO模型）中开展核纤层蛋白B1（LaminB1）染色质免疫共沉淀测序（ChIP-seq），探究缺失依赖H3K9me3的异染色质时核纤层关联的破坏情况；其中Suv39h1与Suv39h2是催化哺乳动物异染色质H3K9me3沉积的主要组蛋白甲基转移酶。实验整体设计：本研究分析对象为CD4+CD8+双阳性小鼠胸腺细胞。分别从Suv39h1+/y Suv39h2+/-（对照）小鼠与Suv39h1-/y Suv39h2-/-双重敲除（Suv39DKO）小鼠各获取两份生物学重复样本，独立制备染色质免疫共沉淀（ChIP）与全细胞提取物（WCE）文库并进行高通量测序，以验证免疫沉淀实验的有效性。生物信息学分析环节，先将两份重复的FASTQ测序文件进行合并；随后分别以敲除组与对照组的WCE数据作为输入对照，调用分析工具识别LaminB1结合峰。