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:
    9098702
  • 负责人:
  • 金额:
    $ 33.3万
  • 依托单位:
  • 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

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(申请人提供):在包括细胞尺寸减小、血红蛋白积累和最终去核的染色质凝聚的终末红细胞发育的过程中,不可逆的染色质凝聚的离散过程仍然知之甚少。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改进治疗的发展,这些贫血的特征是红系成熟晚期缺陷。在原代小鼠红细胞中敲除高度红系特异性的核蛋白Xpo7可导致染色质凝聚和去核的严重中断,但对血红蛋白的积累或红系表达程序几乎没有影响(审稿中),这提供了核蛋白输出对红系染色质凝聚和去核是必不可少的证据。这项提案中概述的工作旨在了解Xpo7从红细胞核输出哪些蛋白质,以及它们如何协同工作来调节红细胞染色质凝聚过程。根据对红系前体核从早期红细胞到挤出的蛋白质组学研究,还注意到挤出的核在很大程度上耗尽了蛋白质--尤其是核心结构蛋白和组蛋白--而缺乏Exportin Xpo7的核积累了几乎所有的非特异性核蛋白。值得注意的是,在去核之前和去核过程中,DNA结合蛋白如组蛋白H2A和H3在正常晚期红细胞的细胞质中积累,但在缺乏Xpo7的细胞中不积累。为了阐明这样一种假设,即Xpo7非特异性地去除染色质缩合的抑制物或所有红系核蛋白,以允许适当的缩合进行适当的去核,本提议旨在了解(1)Xpo7从红系前体核输出哪些蛋白质,以及(2)这些蛋白质如何帮助调节红系染色质缩合。由于固缩、挤压的细胞核中残留的少数蛋白质可能促进了凝聚本身的过程,拟议的工作也将阐明组蛋白蛋白在挤压前如何在红系细胞核中被取代。拟议的研究将依赖于更详细的蛋白质组学和层析,然后是最先进的同时显微镜和流式细胞仪评估,以分别量化染色质凝聚和去核,在体外敲除假定的调节因子。这项研究还将在原代人类细胞培养模型和体内小鼠基因敲除模型中检验这些发现的保守性。这项研究中提出的实验结果也将更详细地揭示Xpo7的功能-确定其功能需要哪些Xpo7特异性基序将有助于解释核出口蛋白如何在没有底物特异性的情况下出口核蛋白。这些知识将增加我们对红系终末成熟过程中核输出和核凝聚等重要基本细胞生物学概念的理解。

项目成果

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Shilpa Manohar Hattangadi其他文献

Shilpa Manohar Hattangadi的其他文献

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{{ truncateString('Shilpa Manohar Hattangadi', 18)}}的其他基金

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:
    8754807
  • 财政年份:
    2014
  • 资助金额:
    $ 33.3万
  • 项目类别:
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
  • 财政年份:
    2014
  • 资助金额:
    $ 33.3万
  • 项目类别:
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:
    9312253
  • 财政年份:
    2014
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    7985263
  • 财政年份:
    2009
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    8063990
  • 财政年份:
    2007
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    7822701
  • 财政年份:
    2007
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    7653613
  • 财政年份:
    2007
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    7179016
  • 财政年份:
    2007
  • 资助金额:
    $ 33.3万
  • 项目类别:
Transcriptional Regulatory Networks Active in Terminal Erythroid Differentiation
转录调控网络在终末红细胞分化中活跃
  • 批准号:
    7433754
  • 财政年份:
    2007
  • 资助金额:
    $ 33.3万
  • 项目类别:

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