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 目前，在美国，造血干细胞（HSC）移植> 16,000次 治疗血液学疾病，白血病并恢复癌症患者的造血。因此，HSC是 大多数临床开发的干细胞群体。最近的多项研究表明造血 骨髓移植后恢复造血的茎和祖细胞（HSPC）是 与维持本地造血的人不同。这些发现当务之急 了解HSPC重生的选择性调节。因此，我们的实验室试图剖析细胞 和调节HSC重生的分子机制。在耗尽基因的功能屏幕中 perturbs HSC在体内重生中，我们确定了核因子I（NFI）基因家族成员NFIX为A BMT后HSPC功能的新型调节剂。 NFIX的损失严重削弱了HSPC重新构成的能力 消融的老鼠。此外，缺乏NFIX的HSPC显示出BMT后增加凋亡，缺乏CFU潜力，并且是 受体骨髓的数量减少。由于多个凋亡调节剂的表达受到干扰 在HSPC中NFIX的存在和不存在的情况下，我们建议NFIX是HSPC存活的关键固有调节剂 BMT。在这里，我们将检验该假设，并评估NFIX在本地造血中的作用。 以下具体目的：1）检验Nfix调节HSPC生存后BMT的假设，2） 确定HSPC中NFIX的分子靶标，以及3）测试在天然造血期间是否需要NFIX。 对于AIM 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凋亡是否至关重要 损失NFIX缺陷后HSPC后BMT。由于缺乏NFIX的HSPC中BCl-XL下调，我们也将 测试如果恢复的BCL-XL表达挽救了缺乏NFIX缺陷HSPC的再现缺陷。最后，nfix 过表达大大延长了原发性造血细胞的离体培养。因此，我们还将测试是否 NFIX过表达可保护HSPC免受体内凋亡。对于AIM 2，我们将利用 HSC缺乏原始造血的NFIX，HSC过表达NFIX和NFIX芯片seq结合模式 细胞系以识别NFIX下游目标的候选目标。这些候选人将在功能上通过 HSPC中ShRNA介导的基因敲低过表达的NFIX在促进NFIX- 这些文化的依赖生存。我们预测，这里必要的基因也将发挥关键作用 - HSPC后BMT中的NFIX流。最后，在AIM 3中，我们将分析NFIX+/+，NFIXFL/+和NFIXFL/FLROSA26CRE-ERT2 骨髓嵌合体用于血统，HSPC骨髓室和HSC的扰动 nfix删除后的静止。他莫昔芬有效地从造血中删除NFIX 这些小鼠的隔室。通过定义NFIX作为HSC监管机构的角色，我们将提高对 HSC并确定可针对BMT后可以增强骨髓植入的新途径。