Mapping the organization of the peripheral nuclear compartment

 描述（由申请人提供）：核内区室对染色体结构和功能做出重要贡献。一个这样的隔室是由核纤层（NL）组织的，核纤层是一种蛋白质网络，在后生动物细胞中排列在核膜（NE）的核质表面。NL包含与大量跨膜蛋白和外周蛋白相关的核纤层蛋白的聚合物组装体。除了作为NE的结构框架外，NL还调节许多信号传导和转录途径，并为核周围的染色质提供束缚和调节位点。核纤层和相关的外周异染色质可被视为包含独特的核区室，本文称为核纤层/外周染色质（NL/PC）区室。虽然已经描述了NL的各个组成部分，但分析并不全面。此外，方法是欠发达的定义其组成变化在细胞分化过程中，并阐明蛋白质-蛋白质相互作用，指定NL网络及其染色质相互作用。为了解决这些基本问题，该项目旨在开发从间充质干细胞（MSC）以及从这些细胞分化的肌细胞和脂肪细胞中分离富集在NL/PC隔室中的亚核组分的方法。随着这些馏分的使用，系统质谱（MS）方法将被开发来表征NL/PC室的蛋白质组成，并定量绘制其在分化过程中的变化。化学交联（XL）与MS相结合将用于绘制组成蛋白质的相互作用，生物化学方法与超分辨率光学显微镜将用于验证这些关联。通过实施这套互补的方法，该项目将提供一个全面的参考图谱，说明NL及其在确定的MSC谱系分化过程中所经历的变化。这将有助于制定关于NL/PC隔室功能的更一般概念，并将确定未来功能研究中可以针对的新组件和节点。这有望大大推进我们对NL在外周异染色质和基因表达的细胞类型特异性调节中的作用的理解。

 DESCRIPTION (provided by applicant): Intranuclear compartments make essential contributions to chromosome structure and function. One such compartment is organized by the nuclear lamina (NL), a protein meshwork that lines the nucleoplasmic surface of the nuclear envelope (NE) in metazoan cells. The NL contains a polymeric assembly of nuclear lamins associated with a large number of transmembrane and peripheral proteins. In addition to serving as a structural framework for the NE, the NL modulates numerous signaling and transcriptional pathways, and provides a tethering and regulatory site for chromatin at the nuclear periphery. The nuclear lamina and associated peripheral heterochromatin can be viewed as comprising a distinctive nuclear compartment, herein termed the nuclear lamina/peripheral chromatin (NL/PC) compartment. Although various components of the NL have been described, the analysis has not been comprehensive. Moreover, approaches are underdeveloped for defining its compositional changes during cell differentiation, and for elucidating the protein-protein interactions that specify the NL network and its chromatin interactions. To address these fundamental issues, this project aims to develop methods to isolate sub-nuclear fractions enriched in the NL/PC compartment from mesenchymal stem cells (MSCs), and from myocytes and adipocytes differentiated from these cells. With the use of these fractions, systematic mass spectrometry (MS) methods will be developed to characterize the protein composition of the NL/PC compartment and to quantitatively chart its changes during differentiation. Chemical crosslinking (XL) combined with MS will be deployed to map the interactions of constituent proteins, and biochemical methods in tandem with super-resolution light microscopy will be used to validate these associations. From implementation of this complementary set of approaches, the project will provide a comprehensive reference map of the NL and the changes that it undergoes during differentiation of defined MSC lineages. This will potentiate the formulation of more general concepts on the functions of the NL/PC compartment, and will identify new components and nodes that can be targeted in future functional studies. This is expected to substantially advance our understanding of the role of the NL in cell-type-specific regulation of peripheral heterochromatin and gene expression.