Elucidating the Role of Nuclear Protein Export in Erythroid Nuclear Condensation

阐明核蛋白输出在红细胞核凝聚中的作用

• 批准号: 8917214
• 负责人: Shilpa Manohar Hattangadi
• 金额: $ 33.3万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8917214
• 关键词: Affect; American; Anemia; Anemia due to Chronic Disorder; Apoptosis; Binding; Biological; Biological Process; CD34 gene; Cell Culture System; Cell Culture Techniques; Cell Maturation; Cell Nucleus; Cell Size; Cells; Chickens; Chromatin; Chromatography; Congenital dyserythropoietic anemia; Cytoplasm; DNA; DNA Maintenance; DNA-Binding Proteins; Defect; Development; Diamond-Blackfan anemia; Disease; Dysmyelopoietic Syndromes; Erythroblasts; Erythrocytes; Erythroid; Erythropoiesis; Erythropoietin; Evaluation; Excision; Exportins; Fetal Liver; Genetic Transcription; Goals; Health; Hematological Disease; Hemoglobin; Heterochromatin; Histone H2A; Histones; Hormones; Human; In Vitro; Knockout Mice; Knowledge; Laboratories; Light; Mammals; Manuscripts; Microscopy; Modeling; Molecular; Mus; Myelogenous; Nuclear; Nuclear Export; Nuclear Protein; Nuclear Proteins; Physical condensation; Play; Process; Protamines; Proteins; Proteomics; Red Blood Cell Count; Refractory anemias; Role; Sideroblastic Anemia; Spermatids; Structural Protein; Substrate Specificity; Thalassemia; Transfusion; Transplantation; Vertebrates; Work; alternative treatment; base; biological systems; gain of function; improved; in vivo; in vivo Model; inhibitor/antagonist; insight; knock-down; leukemia; loss of function; mouse model; novel; prevent; progenitor; programs; promoter; research study; tool; transcription factor

DESCRIPTION (provided by applicant): Among the processes comprising terminal red cell development including decrease in cell size, accumulation of hemoglobin, and chromatin condensation culminating in enucleation, the discrete process of irreversible chromatin condensation is still poorly understood. Our incomplete understanding of this critical process hinders the development of improved treatments for certain erythropoietin-refractory anemias characterized by defects in late erythroid maturation. Knocking down the highly erythroid-specific nuclear exportin Xpo7 in primary murine erythroblasts resulted in severe disruption of chromatin condensation and enucleation but had little effect on hemoglobin accumulation or the erythroid expression program (manuscript in review), providing evidence that export of nuclear proteins is essential to erythroid chromatin condensation and enucleation. The work outlined in this proposal aims to understand which proteins Xpo7 exports from the erythroblast nucleus and how they work together to regulate the process of erythroid chromatin condensation. Based on proteomic examination of the composition of the erythroid precursor nucleus from early erythroblast to extrusion, it was also noted that extruded nuclei are largely depleted of protein-i particular, core structural proteins and histones-while nuclei lacking the exportin Xpo7 accumulated almost all nuclear proteins nonspecifically. Strikingly, DNA binding proteins such as histones H2A and H3 accumulate in the cytoplasm of normal late erythroblasts prior to and during enucleation but not in cells lacking Xpo7. In order to clarify the hypothesis that Xpo7 removes either inhibitors of chromatin condensation or all erythroid nuclear proteins nonspecifically in order to allow adequate condensation for proper enucleation, this proposal aims to understand (1) which proteins Xpo7 exports from the erythroid precursor nucleus, and (2) how these proteins help to regulate erythroid chromatin condensation. Because the few remaining proteins in pycnotic, extruded nuclei likely facilitate the process of condensation itsel, the proposed work will also shed light on how histone proteins are replaced in the erythroid nucleus prior to extrusion. The proposed studies will rely on more detailed proteomics and chromatography, followed by state of the art simultaneous microscopy and flow cytometric evaluation to quantify chromatin condensation and enucleation, respectively, after in vitro knockdown of putative regulators. This study will also examine the conservation of these findings in a primary human cell culture model and an in vivo mouse knockout model. Results from experiments proposed in this study will also uncover the function of Xpo7 in more detail- determining which motifs specific to Xpo7 are required for its function will help explain how a nuclear export protein can export nuclear proteins without substrate specificity. This knowledge will increase our understanding of such important basic cell biological concepts as nuclear export and nuclear condensation during terminal erythroid maturation.

描述（由申请人提供）：在包括终末红细胞发育的过程中，包括细胞大小减小、血红蛋白蓄积和染色质浓缩（最终导致去核），对不可逆染色质浓缩的离散过程仍知之甚少。我们对这一关键过程的不完全理解阻碍了某些红细胞生成素难治性贫血的治疗方法的发展，这些贫血的特征是红细胞成熟晚期的缺陷。在原代小鼠成红细胞中敲低高度红细胞特异性核输出蛋白Xpo 7导致染色质凝聚和去核的严重破坏，但对血红蛋白积累或红细胞表达程序几乎没有影响（手稿在审查中），提供了核蛋白输出对红细胞染色质凝聚和去核至关重要的证据。本提案中概述的工作旨在了解哪些蛋白质Xpo 7从成红细胞核中输出，以及它们如何共同调节红细胞染色质凝聚的过程。基于对从早期成红细胞到挤出的红系前体细胞核的组成的蛋白质组学检查，还注意到挤出的细胞核在很大程度上耗尽了蛋白质，特别是核心结构蛋白和组蛋白，而缺乏exportin Xpo 7的细胞核非特异性地积累了几乎所有的核蛋白。引人注目的是，DNA结合蛋白如组蛋白H2 A和H3在去核之前和去核期间在正常晚期成红细胞的细胞质中积累，但在缺乏Xpo 7的细胞中不积累。为了阐明Xpo 7非特异性地去除染色质凝聚抑制剂或所有红细胞核蛋白以允许适当的凝聚以进行适当的去核的假设，该提议旨在了解（1）哪些蛋白质Xpo 7从红细胞前体核输出，以及（2）这些蛋白质如何帮助调节红细胞染色质凝聚。由于在致密的、挤压的核中剩下的少量蛋白质可能促进了浓缩本身的过程，因此拟议的工作也将阐明组蛋白在挤压前如何在红细胞核中被替换。拟议的研究将依赖于更详细的蛋白质组学和色谱法，其次是最先进的同步显微镜和流式细胞仪评价，以量化染色质凝聚和去核，分别在体外敲低推定的监管机构。本研究还将研究这些发现在原代人细胞培养模型和体内小鼠基因敲除模型中的保守性。这项研究中提出的实验结果也将更详细地揭示Xpo 7的功能-确定Xpo 7的功能所需的特定基序将有助于解释核输出蛋白如何在没有底物特异性的情况下输出核蛋白。这些知识将增加我们对细胞核输出和核浓缩等重要的基本细胞生物学概念的理解。