ETV6 Represses Inflammatory Response Genes and Regulates HSPC Function During Stress Hematopoiesis in Mice [scRNA-seq]

ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly-understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse harboring an Etv6R355X loss-of-function variant, which represents the mouse equivalent to the T5-associated variant ETV6R359X. All HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice under homeostatic conditions; however, these animals exhibit subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineagesca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution, with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF pathway in Etv6+/+ HSPCs, the mouse BM-progenitor derived HPC5 cell line, and G-CSF-mobilized human CD34+ cells. Further, single-cell RNA sequencing of mouse LSK cells isolated six-weeks post-competitive transplantation reveals upregulation of inflammatory gene pathways. Corroborating these findings, we observe significantly increased production of TNF by Etv6R355X/+ versus Etv6+/+ HSPCs post-transplantation. From these studies, we conclude that ETV6 represses inflammatory response genes within HSPCs under conditions of hematopoietic stress, and that this mechanism may be critical to sustain HSPC function. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse harboring Etv6R355X, the murine equivalent to a T5-associated variant ETV6R359X. Young Etv6R355X/+ mice retained normal frequencies of lineage-sca1+cKit+ (LSK) cells but LSK frequencies were significantly decreased in 12-month-old animals. Single cell RNAseq of post-transplant LSK enriched bone marrow from Etv6R355X/+ and Etv6 +/+ mice.

ETS变体6（ETV6）编码一种必需的转录阻遏物，在造血干细胞和祖细胞（HSPCs）中高表达，该蛋白对于成人造血作用不可或缺。杂合致病性生殖系ETV6变异与血小板减少症5（T5）相关，这是一种致病机制尚不明确的遗传性疾病，可增加血小板减少症及血液系统恶性肿瘤的患病风险。
为阐明生殖系ETV6变异如何影响HSPC库并参与疾病发生，我们构建了携带Etv6R355X功能丧失变异的敲入小鼠，该变异对应于与T5相关的人源变异ETV6R359X。稳态条件下，Etv6R355X/+小鼠骨髓（BM）中的所有HSPC亚群均正常存在；但该类小鼠的特定祖细胞亚群的比例和/或数量出现细微改变。
为检测Etv6R355X/+突变是否影响HSPC功能，我们开展了连续竞争性移植实验，结果发现Etv6R355X/+谱系阴性-sca1阳性-cKit阳性（LSK）细胞的造血重建能力显著受损，在第三次移植后几乎完全无法使受辐照的受体小鼠实现造血重建。
结合CUT&RUN、ATAC测序（ATAC-Seq）及Hi-C染色体构象捕获技术的机制研究显示，在野生型Etv6+/+ HSPCs、小鼠骨髓祖细胞来源的HPC5细胞系，以及粒细胞集落刺激因子（G-CSF）动员的人CD34+细胞中，ETV6均可结合炎症基因位点，包括肿瘤坏死因子（TNF）通路相关基因。进一步对竞争性移植6周后分离的小鼠LSK细胞开展单细胞RNA测序，结果发现炎症基因通路出现上调。与此研究结果相符，我们观察到移植后Etv6R355X/+ HSPCs相较于Etv6+/+ HSPCs，其TNF的分泌量显著升高。
基于上述研究，我们得出结论：在造血应激条件下，ETV6可阻遏HSPCs内的炎症反应基因，这一机制可能对维持HSPC功能至关重要。
为进一步阐明生殖系ETV6变异如何影响HSPC库并参与疾病发生，我们构建了携带Etv6R355X的敲入小鼠，该变异对应于与T5相关的人源变异ETV6R359X。年轻的Etv6R355X/+小鼠的LSK细胞频率保持正常，但12月龄小鼠的LSK细胞频率显著降低。我们对来自Etv6R355X/+及Etv6+/+小鼠的移植后富集LSK的骨髓细胞开展了单细胞RNA测序。