An Intergenic Silencer Region Regulates HBG Expression

The most common genetic disorders worldwide are the ß-hemoglobinopathies, including sickle cell disease and ß-thalassemia caused by HBB gene mutations. Five functional ß-like globin genes on chromosome 11 are expressed during development in a tissue and stage-specific manner. The HBG genes encoding g-globin polypeptide chains to form fetal hemoglobin (HbF), undergo gene silencing during the first year of life. Our study aimed to characterize an upstream DNA regulatory region that can be targeted for HBG reactivation during adult development. We investigated the intergenic region between HBE1 and HBG2 which we termed the fetal chromatin domain (FCD) to define its role in globin gene regulation. Using normal human CD34+ stem cells to establish erythroid progenitors, we characterized the binding of different transcription factors to the FCD, which facilitated long-range chromatin looping between the FCD and locus control region and HBG. Using CRISPR/Cas9 genome editing technology, the FCD was knocked out in KU812 and CD34+ stem cells. We observed enhanced HBG transcription and HbF expression in both lines. For FCD knockout CD34+ stem cells, flow cytometry showed increased HbF positive cells and HbF expression and enhanced long-range interactions between the locus control and HBG region. Single cell RNA-sequencing revealed normal erythropoiesis in the FCD-KO line and partial reversal of HBG silencing. These results support a putative silencer role of the FCD in regulating HBG transcription and HbF expression. Overall design: The FCD knockout or AAVS1 edited controls were generated in CD34+ stem cells by CRISPR/Cas9 in the Hematopoietic Progenitor/Stem Cell Gene Editing core of the Fred Hutchinson Cancer Center. CD34+ stem cells were then grown in primary culture system. and harvested on Day 10 for scRNA-seq. Cells were resuspended in ice-cold cryopreservation solution comprised of 50% fetal bovine serum, 40% growth media, and 10% DMSO and store at -80°C until shipping to Active Motif (Carlsbad, CA). Clustering algorithms are applied to identify distinct cell populations and further characterization of gene expression patterns within these clusters was performed. The resulting data were used to compare gene expression patterns across erythroid cell populations and to identify differentially expressed genes.

全球范围内最常见的遗传疾病为β-血红蛋白病（ß-hemoglobinopathies），涵盖镰状细胞病（sickle cell disease）以及由HBB基因（hemoglobin subunit beta, HBB）突变引发的β-地中海贫血（ß-thalassemia）。11号染色体上存在5个功能性β类珠蛋白基因，在发育过程中以组织特异性和阶段特异性方式表达。编码γ-珠蛋白多肽链以形成胎儿血红蛋白（HbF，fetal hemoglobin）的HBG基因，会在出生后第一年发生基因沉默。本研究旨在鉴定可在成人发育阶段靶向激活HBG基因的上游DNA调控区域。我们对HBE1与HBG2之间的基因间区进行了研究，将其命名为胎儿染色质结构域（FCD，fetal chromatin domain），以明确其在珠蛋白基因调控中的作用。

我们利用正常人CD34+干细胞构建红细胞祖细胞，对不同转录因子与FCD的结合情况进行了表征，该结合过程可介导FCD与位点控制区（locus control region）及HBG之间的长距离染色质环化。借助CRISPR/Cas9基因组编辑技术，我们在KU812细胞系与CD34+干细胞中敲除了FCD。在两种细胞系中均观察到HBG转录水平与HbF表达量升高。针对FCD敲除的CD34+干细胞，流式细胞术检测显示HbF阳性细胞比例及HbF表达量均有所提升，同时位点控制区与HBG区域之间的长距离相互作用增强。单细胞RNA测序（scRNA-seq，single cell RNA-sequencing）结果表明，FCD敲除细胞系的红细胞生成过程正常，且HBG基因沉默现象得到部分逆转。上述结果证实，FCD在调控HBG转录与HbF表达中发挥推定的沉默子功能。

研究整体设计：本研究通过弗雷德·哈钦森癌症中心造血祖细胞/干细胞基因编辑核心平台的CRISPR/Cas9技术，在CD34+干细胞中构建FCD敲除细胞系，并以AAVS1位点编辑作为对照。随后将CD34+干细胞置于原代培养体系中培养，于第10天收获细胞用于单细胞RNA测序。将细胞重悬于由50%胎牛血清、40%生长培养基及10%二甲基亚砜（DMSO）组成的低温冻存液中，置于-80℃储存，直至运送至美国加利福尼亚州卡尔斯巴德的Active Motif公司开展后续实验。通过聚类算法鉴定不同的细胞群体，并对这些群体内的基因表达模式进行进一步表征。所得数据用于比较不同红细胞群体的基因表达特征，并筛选差异表达基因。