Reactivation of Zeta-Globin by deletion of BCL11A and LRF in HUDEP cells (ChIP-seq). Reactivation of Zeta-Globin by deletion of BCL11A and LRF in HUDEP cells (ChIP-seq)

The alpha and beta-globin loci harbour genes that are expressed during development and silenced throughout post-natal life. Learning to reactivate these genes may offer new therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address the mechanisms underlying the regulation of the gene encoding zeta-globin, the embryonically expressed alpha-like globin. Overall design: CRISPR/Cas9 editing was carried out to generate homozygous clones where LRF, BCL11A and BCL11A/LRF were homozygously deleted. Effects of edited variants were determined by ChIP-seq of chromatin marks in technical replicates. Experiments were carried out at Day 10 of differentiation on unsorted cells.

α-珠蛋白基因座与β-珠蛋白基因座携带着在发育阶段表达、而在整个产后生命周期中被沉默的基因。重新激活此类基因的相关研究，可为血红蛋白病（hemoglobinopathies）——最为常见的单基因遗传病——提供全新的治疗策略。本研究旨在解析编码ζ-珠蛋白（zeta-globin）的基因的调控机制，该基因属于胚胎阶段表达的类α珠蛋白家族成员。
整体实验设计：采用CRISPR/Cas9基因编辑技术构建纯合克隆，分别实现LRF、BCL11A的纯合缺失，以及BCL11A与LRF的联合纯合缺失。通过对染色质修饰标记进行染色质免疫共沉淀测序（ChIP-seq）并设置技术重复，以此评估编辑后的变异株的调控效应。所有实验均在未分选的细胞中，于细胞分化第10天开展。