Nuclear organization in stem and differentiated cells

描述（由申请人提供）：该申请涉及广泛的挑战领域（14）干细胞和特定的挑战主题，14- ag -105，来自健康个体和精神/神经系统疾病患者的诱导多能干细胞（iPS）的探索性研究。染色质绝缘体是大的DNA蛋白复合物，在调节基因表达中起作用。这些复合物的结构、组成和功能在果蝇中得到了很好的理解，但在哺乳动物中却知之甚少。我们实验室的工作在很大程度上促成了绝缘体在细胞核功能中的作用的当前观点，即细胞核内染色质纤维的三维组织。现在的一个关键问题是确定这种组织对于驱动细胞分化的基因表达模式的建立和/或维持是否重要。我们提出实验策略来了解干细胞分化过程中控制核组织的分子机制。考虑到诱导多能干细胞对人类健康可能的重要性，了解分化细胞被重新编程为诱导多能干细胞时发生的所有表观遗传变化，包括核组织的变化，也是至关重要的。我们实验室的初步结果表明，小鼠干细胞中CTCF绝缘体的组织随着这些细胞的分化而改变。此外，最近有大量的信息描述了CTCF蛋白在各种细胞类型中的基因组定位。这些基因组CTCF位点之间的相互作用决定了细胞核中DNA的组织模式。在这里，我们建议确定在不同的分化状态的细胞基因组的一个区域的三维结构。这将使用5C来确定CTCF位点子集之间所有可能的相互作用，并使用用于分析NMR蛋白质数据的计算方法来分析数据。5C实验将使用小鼠干细胞、神经祖细胞、神经源性iPS细胞和多巴胺能神经元进行。结果将揭示干细胞、分化细胞和iPS细胞之间的表观遗传差异和相似性。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-AG-105 Exploratory studies of induced pluripotent stem (iPS) cells from healthy individuals and patients with mental/nervous system disorders. Chromatin insulators are large DNA protein complexes that play a role in regulating gene expression. The structure, composition and function of these complexes is well understood in Drosophila but much less information is available in mammals. Work in our laboratory has contributed in large part to the current view of the role of insulators in nuclear function i.e. the three-dimensional organization of the chromatin fiber within the nucleus. A critical issue now is to determine whether this organization is important for the establishment and/or maintenance of patterns of gene expression that drive cell differentiation. We propose experimental strategies to understand the molecular mechanisms controlling nuclear organization during the process of stem cell differentiation. Given the possible importance of iPS cells to human health, it is critical also to understand all epigenetic changes that take place when differentiated cells are reprogrammed into iPS cells, including changes in nuclear organization. Preliminary results in our lab suggest that the organization of CTCF insulators in mouse stem cells changes as these cells differentiate. In addition, a wealth of information has become recently available describing the genomic localization of the CTCF protein in a variety of cell types. Interactions between these genomic CTCF sites determine the pattern of organization of the DNA in the nucleus. Here we propose to determine the three-dimensional structure of a region of the genome in cells at different states of differentiation. This will be accomplished using 5C to determine all possible interactions between a subset of CTCF sites and analyzing the data using computational methods derived from those used to analyze NMR protein data. The 5C experiments will be carried out with mouse stem cells, neural progenitor cells, neural-derived iPS cells and dopaminergic neurons. Results will shed light on the epigenetic differences and similarities between stem, differentiated and iPS cells. PUBLIC HEALTH RELEVANCE: The arrangement of the DNA of in the nucleus of eukaryotic cells is important in the regulation of gene transcription. DNA sequences called insulators and associated proteins are important in establishing the pattern of DNA organization in the nucleus. We propose to study how insulators contribute to changes in DNA organization taking place when stem cells become differentiated cells and when these cells are re-programmed to become iPS cells.