Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening (human_HSC_RNA_Seq)

Mitochondria have recently been identified as a critical regulator for the homeostasis of hematopoietic stem cells (HSCs). However, the mechanism underlying HSC regulation still needs to be clarified. Here, we identify transcription factor Nynrin as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Nynrin is highly expressed in HSC under steady and stress state. Nynrin knockout leads to significantly decreased long-term HSC frequency, markedly reduced HSC dormancy, and self-renewal capacity in steady-state and stress hematopoiesis. We observed abnormal mitochondrial metabolism and mitochondrial permeability transition pore (mPTP) opening in Nynrin-deficient HSCs. Notably, Nynrin-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil-induced stresses and exhibit typical phenotypes of necrosis. In contrast, overexpression of Nynrin in HSC is resistant to the radiation. Mechanistically, Nynrin deletion induces transactivation of Ppif. Overexpression of cyclophilin D (CypD), the protein encoded by the Ppif gene, causes mPTP opening, mitochondrial swelling, ROS overinduction, and cell necrosis. Both blocking the function of CypD by using cyclosporin A (CsA) or reducing the expression of Ppif could inhibit Nynrin deficiency-induced mitochondrial metabolism enhancement and ROS overproduction, thereby evidently rescuing the impairment of HSCs in Nynrin mutant mice. Collectively, our data, for the first time, characterize Nynrin as a critical regulator of HSC function acting through the Ppif/mPTP mitochondria axis and highlight the importance of Nynrin in HSC maintenance. These data provide new insights into the mechanisms for controlling HSC fate.? Overall design: CD34+ cells were enriched from total bone marrow cells accroding to EasySep™ Human Progenitor Cell Enrichment Kit II. TheCD34+ cells werewere irradiated with 2Gy ?-rays. We then performed gene expression profiling analysis using data obtained from RNA-seq of human CD34+ Comparative gene expression profiling analysis of RNA-seq data for nonirradiated CD34+ and irradiated CD34+线粒体近期被证实为造血干细胞（hematopoietic stem cells，HSCs）稳态调控的关键因子。然而，HSC调控的潜在分子机制仍有待阐明。本研究鉴定出转录因子Nynrin是通过调控线粒体功能以维持HSC稳态的新型调控因子。研究发现，Nynrin在稳态及应激状态下的HSC中均呈高表达。Nynrin基因敲除会导致稳态及应激造血过程中，长期HSC频率显著降低，HSC的休眠性与自我更新能力明显受损。我们在Nynrin缺陷型HSC中观察到线粒体代谢异常以及线粒体通透性转换孔（mitochondrial permeability transition pore，mPTP）开放异常。值得注意的是，Nynrin缺陷型HSC对电离辐射及5-氟尿嘧啶（5-fluorouracil）诱导的应激耐受能力更弱，并呈现出典型的坏死表型。与之相反，在HSC中过表达Nynrin则可使其耐受电离辐射。从机制上来说，Nynrin基因缺失会诱导Ppif的反式激活。由Ppif基因编码的亲环蛋白D（cyclophilin D，CypD）过表达会引发mPTP开放、线粒体肿胀、活性氧（reactive oxygen species，ROS）过度生成以及细胞坏死。通过使用环孢菌素A（cyclosporin A，CsA）阻断CypD功能，或是降低Ppif的表达水平，均可抑制Nynrin缺陷诱导的线粒体代谢亢进与ROS过度生成，从而显著挽救Nynrin突变小鼠中HSC的功能损伤。综上，本研究首次证实Nynrin是通过Ppif/mPTP线粒体轴调控HSC功能的关键因子，并阐明了Nynrin在维持HSC稳态中的重要作用。本研究结果为调控HSC命运的分子机制提供了全新的见解。 整体实验设计：我们依照EasySep™ 人类祖细胞富集试剂盒II（EasySep™ Human Progenitor Cell Enrichment Kit II）的操作步骤，从全骨髓细胞中富集CD34阳性（CD34+）细胞。将上述CD34+细胞以2戈瑞（Gy）的剂量进行γ射线辐照。随后我们基于人类CD34+细胞的RNA测序（RNA-seq）数据开展基因表达谱分析，对未辐照与辐照后的CD34+细胞的RNA-seq数据进行对比基因表达谱分析。