p53-Dependent Induction of P2X7 on Hematopoietic Stem and Progenitor Cells Regulates Hematopoietic Response to Genotoxic Stress [ChIP-seq]

Stem and progenitor cells are the main mediators of tissue renewal and repair, both under homeostatic conditions and in the response to physiological stress and injury. Hematopoietic system is responsible for the regeneration of blood and immune cells, and is maintained by bone marrow resident hematopoietic stem and progenitor cells (HSPCs). Hematopoietic system is particularly susceptible to injury in response to genotoxic stress, resulting in risk of bone marrow failure and secondary malignancies in cancer patients undergoing radiotherapy. Here we analyze the in vivo transcriptional response of HSPCs to genotoxic stress in a mouse whole body irradiation model, and together with p53 ChIP-Seq and studies in p53-knockout (p53KO) mice, characterize the p53-dependent and p53-independent branches of this transcriptional response. Our work demonstrates the p53-independent induction of inflammatory transcriptional signatures in HSPCs in response to genotoxic stress, and identifies multiple novel p53-target genes induced in HSPCs in response to whole body irradiation. In particular, we establish the direct p53-mediated induction of P2X7 expression on HSCs and HSPCs in response to genotoxic stress. We further demonstrate the role of P2X7 in hematopoietic response to acute genotoxic stress, with P2X7-deficiency significantly extending mouse survival in irradiation-induced hematopoietic failure. We also demonstrate the role of P2X7 in the context of long-term HSC regenerative fitness following sublethal irradiation. Overall our studies provide important insights into the mechanisms of HSC response to genotoxic stress and further suggests P2X7 as a target for pharmacological modulation of HSC fitness and hematopoietic response to genotoxic injury. To map p53 DNA binding sites, p53 chromatin immunoprecipitation sequencing (ChIP-Seq) was performed in multipotent progenitor cells HPC7 and B cell progenitors Ba/F3, both selected for their p53 wild type status. In parallel, ChIP-Seq was done in Ba/F3s following irradiation (3Gy, 3 hours).

干细胞与祖细胞是稳态条件下及生理应激与损伤应答过程中组织更新与修复的核心介导因子。造血系统负责血液与免疫细胞的再生，并由骨髓驻留的造血干细胞与祖细胞（hematopoietic stem and progenitor cells，HSPCs）维持稳态。造血系统对基因毒性应激尤为敏感，易受损伤，这会使接受放疗的癌症患者面临骨髓衰竭与继发性恶性肿瘤的风险。本研究在小鼠全身照射模型中分析了HSPCs对基因毒性应激的体内转录应答，并结合p53染色质免疫沉淀测序（ChIP-Seq）数据与p53敲除（p53KO）小鼠实验，解析了该转录应答中p53依赖与非依赖两条通路。本研究证实，HSPCs在基因毒性应激下会通过p53非依赖通路诱导炎症相关转录特征，并筛选出多个在全身照射后于HSPCs中被诱导表达的新型p53靶基因。尤为重要的是，本研究明确了基因毒性应激下，p53直接介导造血干细胞（hematopoietic stem cells，HSCs）与HSPCs中P2X7的表达上调。本研究进一步证实了P2X7在造血系统对急性基因毒性应激的应答中的作用：P2X7缺陷可显著延长照射诱导的造血衰竭小鼠的存活时间。此外，本研究还阐明了P2X7在亚致死照射后造血干细胞长期再生能力维持中的功能。综上，本研究为造血干细胞应对基因毒性应激的机制提供了重要见解，并进一步提出P2X7可作为调控造血干细胞功能与造血系统对基因毒性损伤应答的药理学靶点。为绘制p53 DNA结合位点图谱，本研究在p53野生型的多能祖细胞HPC7与B细胞祖细胞Ba/F3中开展了p53 ChIP-Seq实验。同时，本研究还对经3Gy照射3小时后的Ba/F3细胞开展了ChIP-Seq实验。