Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening (RNA-Seq for Nynrin cko HSC)

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. The construct of the whole cDNA library for low number HSCs (500 cells)from Nynrin cko mice was performed as previously described (Chen et al., 2017). In brief, about 3000 HSCs of BMs from Nynrinfl/flTie2-Cre or Nynrinfl/fl mice were sorted by FACS. cDNA was synthesized according to Jun Chen et al. (2017). The mRNA library was constructed using TruePrepTM DNA Library Prep Kit V2 for Illumina (Vazyme) and TruePrepTM Index Kit V2/V3 for Illumina (Vazyme) for sequencing. Libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Each library was quantified using a Qubit fluorometer (Thermo Fisher Scientific), and the size distribution was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). The Illumina HiSeq 2000 platform sequenced libraries as 350-bp pair-ended reads.线粒体近期被证实是造血干细胞（hematopoietic stem cells, HSCs）稳态维持的关键调节因子。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实现HSC稳态维持的新型调节因子。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造血干细胞频率显著降低，HSC的休眠能力与自我更新能力明显受损。在Nynrin缺陷的HSC中，我们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缺陷的HSC对辐照及5-氟尿嘧啶（5-fluorouracil, 5-FU）诱导的应激耐受能力显著下降，并呈现典型的坏死表型。与之相反，在HSC中过表达Nynrin则可使其耐受辐照损伤。从机制层面来看，Nynrin缺失会诱导Ppif基因的转录激活。Ppif基因编码的亲环蛋白D（cyclophilin D, CypD）过表达可引发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。使用环孢菌素A（cyclosporin A, CsA）阻断CypD功能，或是下调Ppif的表达，均可抑制Nynrin缺陷所诱导的线粒体代谢增强与ROS过度产生，从而显著挽救Nynrin突变小鼠中HSC的功能损伤。综上，本研究首次明确Nynrin是通过Ppif/mPTP线粒体轴调控HSC功能的关键调节因子，并揭示了Nynrin在维持HSC稳态中的重要作用。上述研究结果为调控HSC命运的分子机制提供了新的见解。针对来自Nynrin条件性敲除（conditional knockout, cko）小鼠的少量HSC（500个细胞）的全长cDNA文库构建，按照此前报道的方法（Chen等，2017）完成。简言之，我们通过流式细胞术（FACS）分选出约3000个来自Nynrinfl/flTie2-Cre或Nynrinfl/fl小鼠骨髓的HSC。参照Jun Chen等（2017）的方法合成cDNA。mRNA文库构建采用TruePrep™ DNA Library Prep Kit V2 for Illumina（Vazyme）与TruePrep™ Index Kit V2/V3 for Illumina（Vazyme）完成，用于后续测序。文库纯化采用Agencourt AMPure XP磁珠（Beckman Coulter）完成。每个文库通过Qubit荧光定量仪（Thermo Fisher Scientific）进行定量，并使用2100生物分析仪（Agilent Technologies, 美国圣克拉拉）评估片段大小分布。采用Illumina HiSeq 2000平台对文库进行测序，读取模式为350 bp双端reads。