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: 9029369
• 负责人: SHANNON L MCKINNEY-FREEMAN
• 金额: $ 40.11万
• 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/9029369
• 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; Data; Defect; Disease; 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); 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; Stream; System; TNFRSF5 gene; Tamoxifen; Testing; Time; Transplantation; United States; Work; adult stem cell; gene discovery; 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.

项目概要 目前在美国每年使用造血干细胞 (HSC) 移植超过 16,000 次 治疗血液疾病、白血病，并恢复癌症患者的造血功能。因此，HSC 是其中之一 大多数临床利用的干细胞群。最近多项研究表明，造血 骨髓移植（BMT）后恢复造血功能的干细胞和祖细胞（HSPC）是 与维持天然造血功能的细胞不同。这些发现迫切需要更好的 了解 HSPC 增殖的选择性调节。因此，我们的实验室试图剖析细胞 以及调节 HSC 增殖的分子机制。在基因的功能筛选中，其耗尽 扰乱 HSC 体内再增殖，我们确定了核因子 I (NFI) 基因家族成员 Nfix 作为一个 BMT 后 HSPC 功能的新型调节剂。 Nfix 的丢失严重削弱了 HSPC 的重建能力 消融的小鼠。此外，Nfix 缺陷的 HSPC 在 BMT 后表现出细胞凋亡增加，缺乏 CFU 潜力，并且 受体骨髓中的数量减少。由于多种凋亡调节因子的表达受到干扰 在 HSPC 中存在和不存在 Nfix 的情况下，我们认为 Nfix 是 HSPC 存活的关键内在调节因子 骨髓移植后。在这里，我们将测试这一假设，并评估 Nfix 在天然造血中的作用，根据 达到以下具体目标：1) 检验 Nfix 调节 BMT 后 HSPC 存活的假设，2) 确定 HSPC 中 Nfix 的分子靶标，以及 3) 测试天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至关重要 BMT 后 Nfix 缺陷型 HSPC 的丢失。由于 Bcl-xL 在 Nfix 缺陷的 HSPC 中下调，我们还将 测试恢复的 Bcl-xL 表达是否可以挽救 Nfix 缺陷的 HSPC 的重新填充缺陷。最后，N修复 过度表达大大延长了原代造血细胞的离体培养时间。因此，我们还将测试是否 Nfix 过表达可保护 HSPC 免于离体凋亡。对于目标 2，我们将利用以下转录组 原始造血系统中缺乏 Nfix 的 HSC、过表达 Nfix 的 HSC 和 NFIX ChIP-seq 结合模式 细胞系来识别 Nfix 的候选下游靶标。这些候选者将通过以下方式进行功能验证 shRNA 介导的 HSPC 过表达 Nfix 中的基因敲低在促进 Nfix- 这些文化依赖生存。我们预测这里必需的基因也将发挥关键作用： BMT 后 HSPC 中的 Nfix 流。最后，在目标 3 中，我们将分析 Nfix+/+、Nfixfl/+ 和 Nfixfl/flROSA26Cre-ERT2 用于扰动血统、HSPC 骨髓区室和 HSC 的骨髓嵌合体 Nfix 删除后处于静止状态。他莫昔芬治疗可有效消除造血系统中的 Nfix 这些小鼠的隔间。通过定义 Nfix 作为 HSC 监管者的角色，我们将加深对 HSC 并确定可用于增强 BMT 后骨髓移植的新途径。