Genome-wide maps of H3K4Me3 histone modification in human pulmonary endothelial cells under hypoxia

Purpose: In lung tissue of mice with experimental PH, we identified an upregulated lncRNA 5031425E22 which mapped to a human ortholog KMT2E-AS1. Across mammals, this lncRNA gene sits adjacent (head-to-head) to the chromosomal location of the histone lysine N-methyltransferase 2E gene (KMT2E), a member of a family of regulators controlling histone 3 lysine 4 trimethylation (H3K4Me3) and chromatin remodeling. We found that both mouse lncRNA E22 and human KMT2E-AS1 were increased in multiple in vivo rodent and human instances of PH. This ChIP-sequencing study was designed to compare the histone 3 lysine 4 methylated regions/gene profile of human pulmonary arterial endothelial cells (HPAECs) under hypoxia as compared to normoxia. Method: Primary HPAECs were grown in EBM-2 basal medium supplemented with EGM-2 MV BulletKit (Lonza). Experiments were performed at passages 5 to 8. Cultured HPAECs were either under normoxia or hypoxia (0.2% oxygen) for 24 hours. Cells were fixed, crosslinked, and sonicated to get soluble chromatin for immunoprecipitation. 10% input of crosslinked DNA was saved while the remining DNA was precipitated with histone 3 lysine 4 antibody (H3K4Me3, Abcam, ab8580) or non-immune rabbit IgG (Pierce magnetic ChIP kit). Chromatin/antibody complex was then pulled down using Protein A/G Magnetic Beads (Pierece). DNA samples were proceeded with ChIP-sequencing (sequencing platform: DNBSeqTM, 20M reads). Overall design: H3K4Me3 ChIP pulldown of HPAECs under hypoxia vs normoxia using 7 total samples: 1 control, 3 normoxia, and 3 hypoxia.

研究目的：在患有实验性肺动脉高压（PH）的小鼠肺组织中，我们鉴定出一条上调的长链非编码RNA（long non-coding RNA, lncRNA）5031425E22，其人类同源基因为KMT2E-AS1。在哺乳动物类群中，该lncRNA基因与组蛋白赖氨酸N-甲基转移酶2E基因（KMT2E）的染色体位点呈头对头相邻排布；KMT2E属于调控组蛋白3赖氨酸4三甲基化（H3K4Me3）与染色质重塑的调控因子家族成员。我们发现，在多种啮齿类动物与人类的肺动脉高压（PH）体内模型中，小鼠lncRNA E22与人类KMT2E-AS1的表达水平均显著上调。本染色质免疫共沉淀测序（ChIP-sequencing, ChIP-seq）研究旨在对比低氧与常氧条件下人肺动脉内皮细胞（human pulmonary arterial endothelial cells, HPAECs）的组蛋白3赖氨酸4甲基化区域及基因谱差异。
实验方法：原代人肺动脉内皮细胞（HPAECs）采用添加EGM-2 MV BulletKit的EBM-2基础培养基进行培养，实验使用第5至8代细胞。将培养的HPAECs分别置于常氧或低氧（0.2%氧气）环境中培养24小时。随后对细胞进行固定、交联，经超声破碎获取可溶性染色质用于免疫沉淀实验。留存10%的交联DNA作为输入对照，剩余DNA分别使用组蛋白3赖氨酸4三甲基化抗体（H3K4Me3，Abcam，ab8580）或非免疫兔IgG（配套Pierece磁珠ChIP试剂盒）进行沉淀。随后使用Protein A/G磁珠（Pierece）捕获染色质-抗体复合物。对所得DNA样本进行ChIP测序，测序平台为DNBSeqTM，测序数据量为20M读段。
实验整体设计：本研究共纳入7个样本，对低氧与常氧条件下的HPAECs开展H3K4Me3 ChIP富集测序，其中包含1个对照样本、3个常氧组样本与3个低氧组样本。