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

基本信息

批准号：
8754807
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
项目类别：
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/8754807
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 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 public health relevance research study tool transcription factor

项目摘要

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）：在包含末端红细胞发育的过程中，包括细胞大小的降低，血红蛋白的积累和染色质凝结最终导致摘除中的最终，不可逆的染色质冷凝的离散过程仍然很少了解。我们对这一关键过程的不完全理解阻碍了对某些以骨膜成熟后缺陷为特征的某些促红细胞生成素 - 杀情性贫血的治疗的发展。 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.该提案中概述的工作旨在了解哪些蛋白质XPO7从红细胞核中导出了哪些蛋白质，以及它们如何共同调节红细胞染色质凝结的过程。基于蛋白质组学检查从早期红细胞到挤出的红细胞前体核的组成，还注意到挤出的核在很大程度上耗尽了蛋白质I-i的特定核心结构蛋白和组织蛋白蛋白质 - 丝蛋白 - 丝蛋白 - 缺乏Exportin Xpo7 xpo7 xpo7 xpo7 xpo7 xpo7 xpo7 xpoe xpoe xpoe xpoey xpoe蛋白几乎累积了所有核蛋白质。引人注目的是，在摘除核酸酯之前和期间，DNA结合蛋白（例如组蛋白H2A和H3）在正常的riNTHROMBLASTS的细胞质中积累，而在缺乏XPO7的细胞中则积累。为了阐明XPO7消除染色质凝结抑制剂或所有红斑核蛋白的抑制剂的假设，以便允许适当的凝结以进行适当的摘要，该建议旨在了解（1）哪种蛋白质XPO7从erythroid chrotorid chrotorid chrotorid controuits chrotic controult controuts和2）凝结。由于甲状腺挤出核中的几个剩余蛋白可能促进了凝结的过程，因此拟议的工作还将阐明在挤出之前如何在红细胞核中取代组蛋白蛋白。拟议的研究将依靠更详细的蛋白质组学和色谱法，然后同时进行最先进的显微镜和流式细胞仪评估，以分别量化染色质凝结和摘除剂，在体外敲低推定调节剂后。这项研究还将在原发性的人类细胞培养模型和体内小鼠敲除模型中检查这些发现的保护。本研究中提出的实验的结果还将发现XPO7的功能，更详细地确定其功能所需的特定于XPO7的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导出核蛋白。这些知识将增加我们对末端红斑成熟期间核出口和核凝结等重要基本细胞生物学概念的理解。