Regulation of alpha-like globin gene expression in murine primitive erythropoiesis (NG CaptureC)

Haemoglobin, the oxygen-carrying molecule of red blood cells, at all stages of development comprises a tetramer of two a-like and two ß-like globin chains. In humans and mice the highly conserved a- and ß-globin loci contain genes encoding globin chains specifically expressed only during the first trimester of gestation (termed embryonic globins) in addition to the genes encoding globin chains expressed throughout adult life. In humans the ß-globin locus also encodes a ß-like chain that is expressed throughout the second and third trimesters of development; this is termed ?-globin and complexes with a-globin to form fetal haemoglobin (HbF). The sequential activation and repression of the genes at each of these loci throughout development to maturity is termed haemoglobin switching and is a poorly understood process. a-thalassemia, which results from insufficient production of a-globin, is a major global health problem with several hundred thousand sufferers world-wide. The embryonically expressed ?-globin, encoded by the gene HBZ (Hba-x in mice), can functionally substitute for a-globin in adult erythroid cells. Reactivation of this gene is consequently likely to ameliorate the symptoms of a-thalassemia in individuals with a severe phenotype. Little is currently known about the regulation of ?-globin expression in terms of both the trans- and cis-acting regulatory elements responsible for high-levels of expression of this gene during embryonic erythropoiesis and its maintenance in a transcriptionally inactive state throughout adult life. Using ATACseq and NG Capture C and mutant mouse lines this work aims to identify and define the cis-acting regulatory network underlying zeta-globin expression, and define the contribution of each regulatory element. Overall design: NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments. The method was applied to biological triplicates of embryonic stem cells (ESC), primitive erythroid cells (E10.5), and definitive erythroid cells (Fetal Liver: FL) capturing either promoter or enhancer elements.

血红蛋白（Haemoglobin）是红细胞的携氧分子，在发育的各个阶段均由两条类α与两条类β珠蛋白链构成的四聚体组成。在人类与小鼠中，高度保守的α-与β-珠蛋白基因座，除了编码在成年生命全程表达的珠蛋白链的基因之外，还包含仅在妊娠第一孕期特异性表达的珠蛋白链编码基因，这类珠蛋白被称为胚胎型珠蛋白。在人类中，β-珠蛋白基因座还编码一条在发育的第二、第三孕期全程表达的β样链，该链被称为ζ-珠蛋白（zeta-globin），可与α-珠蛋白结合形成胎儿血红蛋白（HbF）。上述两类基因座在从发育到成熟的全过程中，各基因的依次激活与沉默被称为血红蛋白转换（haemoglobin switching），该过程的分子机制目前仍未阐明。
α-地中海贫血（α-thalassemia）是因α-珠蛋白生成不足所引发的疾病，为全球性重大健康问题，全球患者多达数十万。由HBZ基因（小鼠中为Hba-x）编码的胚胎型ζ-珠蛋白，可在成年红系细胞中功能性替代α-珠蛋白。因此，重新激活该基因有望缓解重度α-地中海贫血患者的临床症状。
目前，关于ζ-珠蛋白表达的调控机制，无论是调控该基因在胚胎红系生成（embryonic erythropoiesis）过程中实现高水平表达的反式作用与顺式作用调控元件，还是维持其在成年生命全程转录沉默的调控元件，相关研究都十分匮乏。本研究将借助ATAC-seq、NG Capture C技术以及突变小鼠模型，旨在鉴定并阐明调控ζ-珠蛋白表达的顺式作用调控网络，并明确各调控元件的具体贡献。
实验整体设计：NG Capture C技术将3C文库制备与针对目标酶切位点的寡核苷酸捕获技术相结合。本研究将该方法应用于胚胎干细胞（ESC）、原始红系细胞（E10.5）以及定型红系细胞（胎肝：FL）的生物学重复样本各三份，分别捕获启动子或增强子元件。