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

项目摘要 造血干细胞（HSC）移植目前在美国使用> 16，000次/年， 治疗血液病、白血病，恢复癌症患者的造血功能。因此，HSC是 临床上最常用的干细胞群。最近的多项研究表明， 在骨髓移植（BMT）后恢复造血干细胞和祖细胞（HSPC）， 与维持天然造血的细胞不同。这些发现使我们有必要更好地 理解HSPC再增殖的选择性调节。因此，我们的实验室试图解剖细胞 以及调控HSC再增殖的分子机制。在一个功能性筛选中， 干扰HSC在体内的再增殖，我们确定核因子I（NFI）基因家族成员，Nfix，作为一个 BMT后HSPC功能的新调节剂。Nfix的丢失严重削弱了HSPC的重建能力 切除的小鼠此外，Nfix缺陷型HSPC在BMT后显示出增加的凋亡，缺乏CFU潜力，并且在BMT后表现出增加的细胞凋亡。 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， 以下具体目的：1）检验Nfix调节BMT后HSPC存活的假设，2） 鉴定HSPC中Nfix的分子靶标，和3）测试在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是关键的 与骨髓移植后Nfix缺陷型HSPC的丢失有关。由于Bcl-xL在Nfix缺陷型HSPC中下调，我们还将 测试恢复的Bcl-xL表达是否挽救了Nfix缺陷型HSPC的再增殖缺陷。最后，Nfix 过表达极大地阻碍了原代造血细胞的离体培养。因此，我们也将测试，如果 Nfix过表达保护HSPC免于离体凋亡。对于目标2，我们将利用以下转录组： 原始造血干细胞中缺乏Nfix的HSC、过表达Nfix的HSC和NFIX ChIP-seq结合模式 细胞系以鉴定Nfix的候选下游靶标。这些候选人将通过以下方式进行功能验证 在过表达Nfix的HSPC中，shRNA介导的基因敲低在促进Nfix- 这些文化的生存。我们预测这里所需的基因也将在下面发挥关键作用- HSPC后BMT中Nfix流。最后，在目标3中，我们将分析Nfix+/+、Nfixfl/+和Nfixfl/flROSA 26 Cre-ERT 2 用于血液谱系、HSPC骨髓区室和HSC中扰动的骨髓嵌合体 Nfix删除后的静止。用他莫昔芬治疗有效地从造血干细胞中删除Nfix。 在这些老鼠身上。通过定义Nfix作为HSC监管机构的角色，我们将提高对以下方面的理解： HSC和确定新的途径，可以有针对性地提高骨髓移植后的骨髓移植。

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.