Mapping the organization of the peripheral nuclear compartment

 描述(申请人提供)：核内室对染色体结构和功能有重要贡献。其中一个隔室是由核层(NL)组成的，核层是一种蛋白质网络，排列在后生动物细胞的核膜(NE)的核质表面。NL含有与大量跨膜和外周蛋白相关的核层蛋白的聚合体。除了作为NE的结构框架外，NL还调节许多信号和转录途径，并为核周的染色质提供一个拴系和调节位置。核层和相关的外周异染色质可以被视为包括一个独特的核室，这里称为核层/外周染色质(NL/PC)室。虽然描述了民族解放力量的各个组成部分，但分析并不全面。此外，在定义其在细胞分化过程中的组成变化，以及阐明指定NL网络及其染色质相互作用的蛋白质-蛋白质相互作用方面，方法还不够成熟。为了解决这些基本问题，本项目旨在开发从间充质干细胞(MSCs)中分离富含NL/PC区的亚核部分，以及从这些细胞分化出的肌细胞和脂肪细胞中分离出亚核部分。随着这些组分的使用，将发展系统质谱学(MS)方法来表征NL/PC隔室的蛋白质组成，并定量地绘制其在分化过程中的变化。化学交联法(XL)将与MS相结合用于绘制组成蛋白质之间的相互作用图，生化方法将与超分辨率光学显微镜相结合用于验证这些联系。通过实施这套相辅相成的方法，该项目将提供一份关于民族解放阵线的全面参考图，以及它在区分已定义的MSC谱系期间所经历的变化。这将加强对NL/PC隔间功能的更一般概念的表述，并将确定可在未来功能研究中作为目标的新组件和节点。这有望极大地促进我们对NL在外周异染色质和基因表达的细胞类型特异性调控中的作用的理解。

 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.