ChIP-seq for E4BP4 in the liver from transgenic mouse with liver specific E4BP4 overexpression. ChIP-seq for E4BP4 in the liver from transgenic mouse with liver specific E4BP4 overexpression

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate the effects of liver-specific E4BP4 overexpression under mouse albumin promoter on the liver glucose and lipid metabolism. Methods: We generated transgenic mice (TG) with liver-specific E4BP4 overexpression, and harvested two livers with one TG mouse each. After cervical dislocation, mouse liver was perfused with a buffer containing Hanks' balanced salt solution, and cross-inked for 30 minutes with 2mM DSG and for 10 minutes with 1% formaldehyde by perfusion, followed by frozen at -80C. Then, Liver was homogenated by gentleMACS Dissociators. Nuclei were isolated and protein-DNA complexes were incubated with antibodies against two kinds of E4BP4 and immunoprecipitated with IgG paramagnetic beads. Results: There were many significant peaks at the promoter/enhancer or intronic region of many genes. Conclusions: E4BP4 possibly regulates the expression of some genes linked to lipid metabolism in the liver. Overall design: ChIP-Seq in liver from transgenic mouse with liver specific E4BP4 overexpression

研究目的：下一代测序（Next-generation sequencing, NGS）彻底革新了基于系统生物学的细胞通路分析。本研究旨在评估小鼠白蛋白启动子驱动的肝脏特异性E4BP4过表达对小鼠肝脏糖脂代谢的影响。
实验方法：我们构建了肝脏特异性过表达E4BP4的转基因小鼠（Transgenic mice, TG），每只转基因小鼠采集两枚肝脏样本。经颈椎脱位处死后，以含汉克斯平衡盐溶液（Hanks' balanced salt solution）的缓冲液灌注小鼠肝脏，先后通过灌注法用2mM二琥珀酰亚胺基辛二酸酯（DSG）交联30分钟、1%甲醛交联10分钟，随后置于-80℃冷冻保存。之后使用gentleMACS组织解离仪对肝脏组织进行匀浆。分离细胞核后，将蛋白质-DNA复合物与针对两种E4BP4的抗体孵育，再使用IgG磁珠进行免疫沉淀。
实验结果：在众多基因的启动子/增强子区域及内含子区域中存在大量显著富集峰。
研究结论：E4BP4可能调控肝脏内与脂代谢相关的部分基因的表达。
整体实验设计：对肝脏特异性过表达E4BP4的转基因小鼠的肝脏样本开展染色质免疫共沉淀测序（ChIP-Seq）