STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia [scRNA-Seq]

Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2 but not its paralogue STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, epigenetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression and local chromatin activation which were not compensated by STAG1. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. We performed complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs). We detected effects overlapping STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis. To determine the impact of stable genomic STAG2 deletion on the cellular differentiation stages of hematopoietic cells, we performed RNA-seq on primary CD34+ cord-blood derived HSPC cultures of independent donors electroporated with sgRNA Cas9 ribonucleoprotein complexes targeting the STAG2 gene (SA2KO). As controls, cells were transfected with a non-targeting guide RNA (NC) Cas9 complex. SA2KO and NC cells of the same donor were stained with two different DNA-barcoded Hashtag antibodies and pooled for 10X GENOMICS single cell transcriptomic library generation. *************************************************************** Due to patient privacy concerns, raw sequencing data of adult AML patients is deposited in the European Genome Archive (EGA) under study number EGAS00001007405. Due to special legal and ethical privacy concerns regarding newborns, raw sequencing data of cord blood HSPC samples cannot be deposited in any online repository. ***************************************************************

黏连蛋白（cohesin）可塑造染色质三维结构，包括增强子-启动子相互作用。其组成亚基尤其是STAG2而非其旁系同源基因STAG1，在髓系恶性肿瘤中频发突变。为阐明白血病发生的潜在机制，我们对急性髓系白血病（acute myeloid leukemia, AML）患者样本的遗传、表观遗传、转录及染色质构象变化进行了全面表征。特定基因座出现了黏连蛋白结合占据、基因表达及局部染色质激活的改变，且无法被STAG1代偿。这些改变可与黏连蛋白突变型AML中被破坏的空间染色质环相关联。我们在原代人造血祖细胞（hematopoietic progenitors, HSPCs）中分别对STAG2或STAG1进行了靶向耗竭。经STAG2敲低后，我们检测到与STAG2突变型AML特异性改变相重合的生物学效应，而STAG1耗竭则未引发此类效应。STAG2缺陷型HSPCs表现出分化能力受损，并维持造血祖细胞样基因表达谱。本研究确立了STAG2作为造血系统中染色质相互作用、基因表达及分化的关键调控因子，并鉴定出可能参与人类白血病发生的候选靶基因。为确定基因组STAG2稳定缺失对造血细胞分化阶段的影响，我们对来自独立供体的CD34阳性脐带血来源HSPC培养物进行了处理：通过电转染靶向STAG2基因的sgRNA Cas9核糖核蛋白复合物（SA2KO组），对照组则转染非靶向向导RNA（NC）Cas9复合物。将同一供体的SA2KO组与NC组细胞用两种不同DNA条形码标记的Hashtag抗体染色后混合，用于10X基因组学（10X GENOMICS）单细胞转录组文库构建。
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受患者隐私保护限制，成人AML患者的原始测序数据已存入欧洲基因组档案库（European Genome Archive, EGA），研究编号为EGAS00001007405。由于新生儿相关特殊法律与伦理隐私问题，脐带血HSPC样本的原始测序数据无法存入任何在线数据库。
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