Elucidating the Role of Nuclear Protein Export in Erythroid Nuclear Condensation

描述（由申请人提供）：在终末红细胞发育的过程中，包括细胞大小的减小，血红蛋白的积累和染色质凝聚最终导致去核，不可逆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促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细胞晚成熟缺陷。敲除小鼠原代红母细胞中高度红细胞特异性的核输出蛋白Xpo7可导致染色质浓缩和去核的严重破坏，但对血红蛋白积累或红细胞表达程序几乎没有影响，这证明核蛋白的输出对红细胞染色质浓缩和去核至关重要。这项研究旨在了解Xpo7从红母细胞核输出哪些蛋白，以及它们如何共同调节红染色质凝聚过程。通过对早期红母细胞至挤压期红系前体核组成的蛋白质组学检查，我们也注意到挤压期细胞核大量缺乏蛋白质，特别是核心结构蛋白和组蛋白，而缺乏输出蛋白Xpo7的细胞核则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H2A和H3在正常晚期红母细胞去核前和去核期间的细胞质中积累，而在缺乏Xpo7的细胞中却没有。为了澄清Xpo7非特异性去除染色质凝聚抑制剂或所有红系核蛋白以允许适当的去核充分凝聚的假设，本提案旨在了解(1)哪些蛋白质Xpo7从红系前体核输出，以及(2)这些蛋白质如何帮助调节红系染色质凝聚。由于缩缩细胞核中剩余的少量蛋白质可能促进了缩缩过程本身，因此提出的工作也将阐明在挤压前红系细胞核中组蛋白是如何被取代的。拟议的研究将依赖于更详细的蛋白质组学和层析，其次是最先进的同步显微镜和流式细胞术评估，分别量化在体外敲除假定的调节因子后的染色质凝聚和去核。本研究还将检验这些发现在原代人类细胞培养模型和小鼠体内敲除模型中的保存性。本研究中提出的实验结果也将更详细地揭示Xpo7的功能——确定哪些特定于Xpo7的基序是其功能所必需的，这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一些重要的基本细胞生物学概念的理解，如红细胞成熟末期的核输出和核凝聚。

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.