ETV6 represses Tumor Necrosis Factor During Stress Hematopoiesis to Regulate Mouse Stem Cell Function

At the root of the blood system lies a heterogenous population of hematopoietic stem and progenitor cells (HSPCs), which reside in the bone marrow (BM) and give rise to diverse blood lineages. Maintaining this pool of self-renewing HSPCs is critical to uphold hematopoiesis during period of stress such as bleeding or infection¹. ETS Variant Transcription Factor 6 (ETV6) is a transcriptional repressor that is highly expressed in HSPCs² where it is essential for development and maintenance of the adult hematopoiesis ³. In 2015, our group⁴ and others^5-7 identified germline pathogenic <em>ETV6</em> variants in families with predisposition to B-acute lymphoblastic leukemia (B-ALL) and thrombocytopenia, defining a new genetic syndrome known as Thrombocytopenia 5 (T5). Subsequently, we performed targeted germline <em>ETV6 </em>sequencing of remission blood samples from over 4,000 children with B-ALL and identified germline <em>ETV6</em> variants in ~1% of cases⁷. <em>In vitro</em> studies revealed that ETV6 variant proteins exhibit impaired repressor activity, reduced DNA binding, and aberrant subcellular localization^4,5,7,8. Overall, these studies indicated that T5-associated germline <em>ETV6</em> variants negatively impact the repressor activity of ETV6. In support of this notion, transcriptional profiling of peripheral blood cells from T5 patients has revealed upregulation of interferon response genes⁹. Nevertheless, little remains known about the mechanisms by which ETV6 regulates the HSPC compartment and how T5-associated <em>ETV6</em> variants contribute to disease. To address these questions, we used CRISPR-Cas9 gene editing to generate a novel mouse model harboring a pathogenic heterozygous germline <em>Etv6 </em>variant, R355X, the murine equivalent to the human T5-associated variant R359X¹⁰. Through the comprehensive study of this model, we describe a novel role for ETV6 during aging and regenerative hematopoiesis and show that the heterozygous <em>Etv6</em>^R355X variant impairs HSC function <em>in vitro</em> and <em>in vivo</em>. Using genomic approaches to interrogate mouse and human HSCs, we identify new ETV6 targets, including the gene encoding Tumor Necrosis Factor (TNF) and genes involved in TNF signaling. Further, we show increased TNF production and cell cycling in <em>Etv6^R355X/+</em> mouse HSPCs post-BM transplantation. Finally, we demonstrate that genetic ablation of <em>Tnf</em> restores the long-term potential of <em>Etv6^R355X/+</em> cells in serial replating assays <em>in vitro</em>. Together, these findings provide novel insights into the pathways regulated by ETV6 and demonstrate how a pathogenic variant impacts ETV6 function in the context of hematopoietic stress. All bulk RNAseq, Cut&amp;Run, ATACseq, and Hi-C data has been submitted to in the Gene Expression Omnibus (accession number GSE213597) and Sequence Read Archive (BioProject number PRJNA880871). Due to the journal’s limitation on submitted supplementary data as an Excel file, all post-analysis supplemental data files are deposited in Dryad data repository to be made available with this manuscript.血液系统的核心是异质性造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）群体，这类细胞定居于骨髓（bone marrow, BM），可分化为多种血液细胞谱系。维持这群具备自我更新能力的HSPCs，对于出血、感染等应激状态下的造血功能维持至关重要¹。ETS变异转录因子6（ETS Variant Transcription Factor 6, ETV6）是一类转录抑制因子，在HSPCs中呈高表达状态²，对成体造血系统的发育与稳态维持不可或缺³。2015年，本研究团队⁴与其他多个研究组^5-7分别在携带B型急性淋巴细胞白血病（B-acute lymphoblastic leukemia, B-ALL）易感性与血小板减少症的家族中，鉴定出生殖系致病性ETV6变异，由此定义了一种全新的遗传综合征——血小板减少症5型（Thrombocytopenia 5, T5）。后续我们对超过4000例B-ALL患儿的缓解期血液样本开展了靶向生殖系ETV6测序，在约1%的病例中检测到生殖系ETV6变异⁷。体外（in vitro）研究显示，ETV6变异蛋白的转录抑制活性受损、DNA结合能力下降，且亚细胞定位出现异常^4,5,7,8。综合上述研究结果，T5相关生殖系ETV6变异会负向调控ETV6的转录抑制功能。为验证这一假说，对T5患者外周血细胞的转录组分析显示，干扰素应答基因的表达水平显著上调⁹。然而，目前对于ETV6调控HSPC群体的分子机制，以及T5相关ETV6变异如何参与疾病发生的过程仍所知甚少。为解答上述科学问题，我们借助CRISPR-Cas9基因编辑技术，构建了一株携带致病性杂合生殖系Etv6变异R355X的新型小鼠模型，该变异对应人类T5相关致病变异R359X¹⁰。通过对该模型的系统性研究，我们揭示了ETV6在衰老与再生性造血过程中的全新功能，并证实杂合Etv6^R355X/+变异可在体外（in vitro）与体内（in vivo）环境中损害造血干细胞（hematopoietic stem cell, HSC）的功能。利用基因组学方法分析小鼠与人类HSC，我们鉴定出一批新的ETV6靶基因，包括编码肿瘤坏死因子（Tumor Necrosis Factor, TNF）的基因以及参与TNF信号通路的相关基因。进一步研究发现，骨髓移植后的Etv6^R355X/+小鼠HSPCs中，TNF的产生与细胞周期均出现上调。最后，我们证实，在体外（in vitro）系列再铺板实验中，敲除Tnf可恢复Etv6^R355X/+细胞的长期增殖潜能。综上，本研究为ETV6所调控的信号通路提供了全新的研究视角，并阐明了致病性ETV6变异在造血应激环境下影响其功能的具体机制。所有批量RNA测序（bulk RNAseq）、Cut&Run、ATAC测序（ATACseq）以及Hi-C数据均已提交至基因表达综合数据库（Gene Expression Omnibus, 登录号GSE213597）与序列读取档案库（Sequence Read Archive, 生物项目编号PRJNA880871）。由于期刊对Excel格式补充数据的提交存在限制，所有后期分析的补充数据文件均已上传至Dryad数据仓库，将随本论文同步公开。