ATRX loss couples genome instability at a G-rich repeat to dysregulation of human alpha-globin expression [scRNA-seq]

Germline mutations in the chromatin remodelling protein ATRX cause a severe developmental disorder associated with a-thalassemia. In addition, ATRX is amongst the twenty genes most frequently mutated in cancer. How ATRX mutations alter gene expression remains unclear. Using the a-globin locus as a model, here we show that ATRX deficiency downregulates a-globin in a subset of cells exhibiting DNA damage. A G-rich repeat at the a-globin locus serves as a potential site of G-quadruplex formation and DNA damage. ATRX binds this repeat co-transcriptionally, and its loss increases R-loop accumulation at this site, leading to local DNA damage and transcriptional disruption in cis. Deletion of this repeat abolishes this effect, while targeted DNA damage reinstates it. These findings reveal a mechanism linking ATRX's role in genome stability to transcriptional regulation, and uncover a molecular basis of human genetic disease mediated by a long-range G-rich repeat. Overall design: ATRX was knocked out using CRISPR-Cas9 in CD34+ haematopoietic stem cells (HSCs) derived from healthy donors. These cells were then differentiated into erythroid cells and analysed for gene expression using single-cell RNA sequencing (Smart-seq2).

染色质重塑蛋白ATRX的生殖系突变会引发一种与α地中海贫血（a-thalassemia）相关的严重发育障碍。此外，ATRX是癌症中最常见突变的二十个基因之一。目前，ATRX突变如何改变基因表达的机制仍不明确。本研究以α-珠蛋白基因座（α-globin locus）为模型，发现ATRX缺失会在出现DNA损伤的细胞亚群中下调α-珠蛋白的表达。α-珠蛋白基因座处的G富集重复序列可作为潜在的G-四链体（G-quadruplex）形成位点与DNA损伤诱发位点。ATRX可共转录结合该重复序列，其缺失会增加该位点的R环（R-loop）积累，进而引发局部DNA损伤与顺式转录调控紊乱。删除该重复序列可消除这一效应，而靶向DNA损伤则可恢复该效应。这些发现揭示了一条将ATRX在基因组稳定性中的作用与转录调控相联系的机制，并阐明了由长距离G富集重复序列介导的人类遗传病的分子基础。整体实验设计：利用CRISPR-Cas9技术在来自健康供体的CD34+造血干细胞（hematopoietic stem cells, HSCs）中敲除ATRX基因。随后将这些细胞诱导分化为红细胞系细胞，并通过单细胞RNA测序（Smart-seq2）分析其基因表达情况。