Defining Nfix's role as a novel key regulator of hematopoietic stem cells

定义 Nfix 作为造血干细胞新型关键调节因子的作用

• 批准号: 9198983
• 负责人: SHANNON L MCKINNEY-FREEMAN
• 金额: $ 40.39万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9198983
• 关键词: Adult; Animal Testing; Apoptosis; Apoptotic; B-Lymphocytes; Binding; Bioinformatics; Blood; Bone Marrow; Bone Marrow Transplantation; Cancer Patient; Candidate Disease Gene; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Cessation of life; ChIP-seq; Chimera organism; Clinical; Data; Defect; Embryo; Engraftment; Erythroid; Exhibits; Family member; Frequencies; Gene Expression; Gene Family; Genes; Goals; Growth; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hippocampus (Brain); Impairment; Laboratories; Life; Marrow; Mediating; Molecular; Molecular Target; Mus; Mutation; Myeloid Cells; NFIX gene; Natural regeneration; Pathway interactions; Patients; Pattern; Play; Population; Postdoctoral Fellow; Proteins; Regulation; Regulator Genes; Research Personnel; Role; Stem cells; System; TNFRSF5 gene; Tamoxifen; Testing; Time; Transplantation; United States; Work; gene discovery; granulocyte; improved; in vivo; knock-down; leukemia; novel; nuclear factor 1; overexpression; reconstitution; relating to nervous system; screening; small hairpin RNA; stem; stem cell population; transcriptome

Project Summary Hematopoietic stem cell (HSC) transplantation is currently used >16,000 times/year in the United States to treat hematologic disease, leukemia, and to restore hematopoiesis in cancer patients. HSC are thus one of the most clinically exploited stem cell populations. Multiple recent studies demonstrate that the hematopoietic stem and progenitor cells (HSPC) that restore hematopoiesis following bone marrow transplant (BMT) are distinct from those that sustain native hematopoiesis. These findings place an imperative on better understanding the selective regulation of HSPC repopulation. Thus, our laboratory seeks to dissect the cellular and molecular mechanisms that regulate HSC repopulation. In a functional screen for genes whose depletion perturbs HSC in vivo repopulation, we identified the Nuclear Factor I (NFI) gene family member, Nfix, as a novel regulator of HSPC function post-BMT. Loss of Nfix severely curtailed the ability of HSPC to reconstitute ablated mice. Further, Nfix-deficient HSPC display increased apoptosis post-BMT, lack CFU potential, and are reduced in number in recipient bone marrow. As the expression of multiple apoptotic regulators is perturbed in the presence and absence of Nfix in HSPC, we propose that Nfix is a key intrinsic regulator of HSPC survival post-BMT. Here, we will test this hypothesis and also assess a role for Nfix in native hematopoiesis, according to the following specific aims: 1) to test the hypothesis that Nfix regulates HSPC survival post-BMT, 2) to identify the molecular targets of Nfix in HSPC, and 3) to test if Nfix is required during native hematopoiesis. For Aim 1, we will employ mice deficient in key cell death pathway regulator genes to test if apoptosis is critical to the loss of Nfix-deficient HSPC post-BMT. As Bcl-xL is downregulated in Nfix-deficient HSPC, we will also test if restored Bcl-xL expression rescues the repopulating defect of Nfix-deficient HSPC. Finally, Nfix overexpression greatly prolongs the ex vivo culture of primary hematopoietic cells. Thus, we will also test if Nfix overexpression protects HSPC from apoptosis ex vivo. For Aim 2, we will leverage the transcriptomes of HSC lacking Nfix, HSC overexpressing Nfix, and NFIX ChIP-seq binding patterns in primitive hematopoietic cell lines to identify candidate downstream targets of Nfix. These candidates will be functionally validated via shRNA-mediated gene knockdown in HSPC overexpressing Nfix for an obligate role in promoting the Nfix- dependent survival of these cultures. We predict that genes necessary here will also play a key role down- stream of Nfix in HSPC post-BMT. Finally, in Aim 3, we will analyze Nfix+/+, Nfixfl/+, and Nfixfl/flROSA26Cre-ERT2 bone marrow chimeras for perturbations in blood lineages, HSPC bone marrow compartments, and HSC quiescence following Nfix deletion. Treatment with tamoxifen efficiently deletes Nfix from the hematopoietic compartment in these mice. By defining Nfix's role as an HSC regulator, we will improve our understanding of HSC and identify new pathways that can be targeted to enhance marrow engraftment after BMT.

项目摘要 造血干细胞(HSC)移植目前在美国每年使用16,000次，以 治疗血液病、白血病，恢复癌症患者的造血功能。因此，HSC是 大多数临床使用的干细胞种群。最近的多项研究表明，造血剂 骨髓移植(BMT)后恢复造血的干细胞和祖细胞(HSPC)是 有别于那些维持天然造血的。这些发现使更好的工作成为当务之急 了解HSPC再种群的选择性调控。因此，我们的实验室试图解剖细胞 以及调节HSC重新繁殖的分子机制。在功能筛查中寻找其耗尽的基因 扰动HSC体内再繁殖，我们鉴定了核因子I(NFI)基因家族成员Nfix是一个 骨髓移植后HSPC功能的新型调节器。失去Nfix严重削弱了HSPC的重组能力 消融的小鼠。此外，缺乏Nfix的HSPC显示骨髓移植后细胞凋亡增加，缺乏CFU潜力，并且 受者骨髓中的数量减少。当多种凋亡调节因子的表达受到干扰时 在HSPC中存在和不存在Nfix的情况下，我们认为Nfix是HSPC生存的关键内在调节因子 在BMT之后。在这里，我们将检验这一假设，并评估Nfix在自然造血中的作用 达到以下具体目的：1)检验NFIX调节骨髓移植后HSPC存活的假设，2) 确定Nfix在HSPC中的分子靶点，以及3)测试在自然造血过程中是否需要Nfix。 对于目标1，我们将使用缺乏关键细胞死亡途径调节基因的小鼠来测试细胞凋亡是否是关键 与骨髓移植后缺乏Nfix的HSPC的丢失有关。由于Bclxl在Nfix缺陷的HSPC中表达下调，我们还将 检测是否恢复了Bclxl的表达，以挽救Nfix缺陷HSPC的再填充缺陷。最后，Nfix 过表达大大延长了原代造血细胞的体外培养时间。因此，我们还将测试是否 NFIX过表达对HSPC体外细胞凋亡的保护作用对于目标2，我们将利用 原始造血系统中缺乏NFIX的HSC、过表达NFIX和NFIX芯片序列结合模式的HSC 以确定NFIX的候选下游靶点。这些候选人将通过以下方式进行功能验证 ShRNA介导的HSPC中高表达Nfix的基因敲除在促进Nfix中的专有作用 依赖于这些文化的生存。我们预测，这里所需的基因也将在未来发挥关键作用- 骨髓移植后HSPC中的Nfix流。最后，在目标3中，我们将分析Nfix+/+、Nfix fl/+和Nfix fl/flROSA26Cre-ERT2 骨髓嵌合体在血统、HSPC骨髓室和HSC中的扰动 删除NFIX后出现停顿。他莫昔芬可有效地将Nfix从造血药中删除 这些老鼠体内的隔间。通过定义NFIX作为HSC监管机构的角色，我们将提高对 并找出可靶向促进骨髓移植后骨髓植入的新途径。