Regulation of alpha-like globin gene expression in murine primitive erythropoiesis (ATAC-seq). Regulation of alpha-like globin gene expression in murine primitive erythropoiesis (ATAC-seq)

Haemoglobin, the oxygen-carrying molecule of red blood cells, at all stages of development comprises a tetramer of two α-like and two β-like globin chains. In humans and mice the highly conserved α- and β-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 β-globin locus also encodes a β-like chain that is expressed throughout the second and third trimesters of development; this is termed γ-globin and complexes with α-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. α-thalassemia, which results from insufficient production of α-globin, is a major global health problem with several hundred thousand sufferers world-wide. The embryonically expressed ζ-globin, encoded by the gene HBZ (Hba-x in mice), can functionally substitute for α-globin in adult erythroid cells. Reactivation of this gene is consequently likely to ameliorate the symptoms of α-thalassemia in individuals with a severe phenotype. Little is currently known about the regulation of ζ-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: ATAC-seq of Primitive and Definitive Erythrocytes (3 replicates each)

血红蛋白（Haemoglobin）是红细胞的携氧分子，在发育的所有阶段均由两条α类珠蛋白链与两条β类珠蛋白链组成的四聚体（tetramer）构成。在人类与小鼠中，高度保守的α珠蛋白基因座与β珠蛋白基因座，除了包含编码成年终生表达的珠蛋白链的基因之外，还含有仅在妊娠第一孕期特异性表达的珠蛋白链编码基因，这类基因被称为胚胎珠蛋白（embryonic globins）。
在人类中，β珠蛋白基因座还编码一种在发育第二、第三孕期全程表达的β样链，即γ珠蛋白（γ-globin），其可与α珠蛋白结合形成胎儿血红蛋白（HbF, fetal haemoglobin）。
在发育至成熟的全过程中，这些基因座上的基因依次发生激活与沉默，这一过程被称为血红蛋白转换（haemoglobin switching），其具体机制仍未被充分阐明。
α地中海贫血（α-thalassemia）是因α珠蛋白生成不足所引发的疾病，属于全球性重大健康问题，全球患者规模达数十万。
胚胎阶段表达的ζ珠蛋白（ζ-globin）由HBZ基因编码（小鼠中对应基因为Hba-x），可在成红细胞（erythroid cells）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所知甚少，既不清楚介导其在胚胎造血（embryonic erythropoiesis）过程中高表达的反式作用调控元件（trans-acting regulatory elements）与顺式作用调控元件（cis-acting regulatory elements），也不明确其在成年后维持转录沉默状态的具体机制。
本研究采用ATAC-seq、捕获C技术（NG Capture C）及突变小鼠模型（mutant mouse lines），旨在鉴定并阐明调控ζ珠蛋白表达的顺式作用调控网络，并明确每个调控元件的具体贡献。
整体实验设计：原始红细胞（Primitive Erythrocytes）与定型红细胞（Definitive Erythrocytes）的ATAC-seq测序（各设置3个生物学重复）