Insulator-mediated chromatin organization during neural lineage commitment

描述（由申请人提供）： 新出现的证据支持染色质绝缘体在高阶染色体环结构的全基因组组织中的核心作用。这项建议的总体目标是研究小鼠胚胎干细胞（ES）分化和发育过程中绝缘子介导的染色质组织的后果。我们的中心假设是，脊椎动物绝缘CTCF分区的基因组到谱系特异性域的共表达基因，促进形成高阶染色质环结构，以响应发展的线索。这一假设将根据三个具体目标进行检验。在目标1中，将利用活细胞共聚焦显微镜来研究CTCF分布和动态真实的时间在ES细胞承诺沿着神经谱系。在目标2中，将使用染色质免疫沉淀结合高通量下一代测序（ChIP-Seq）在ES细胞中鉴定全基因组CTCF结合位点。将多能ES细胞的全局CTCF占用图与多能神经祖细胞和终末分化的神经元进行比较。最后，在目的3中，基于CTCF的染色质环的结构组织将使用在神经谱系定型期间显示差异绝缘子占据的基因组基因座处的染色体构象捕获（3C）来表征。这些CTCF为基础的结构与最近的全基因组分析的“传统的”表观遗传修饰和谱系特异性基因表达谱的相关性将提供一个更全面的了解基因组和表观基因组如何在协调一致，以调节形成一个多样化的阵列组织类型在发展过程中。完成拟议的工作将提供重要的洞察机制，管理的承诺，多能干细胞向神经外胚层谱系。这一知识将使在理解癌症和神经退行性疾病发作期间高阶染色质结构的原因和后果方面取得进展。公共卫生相关性：胚胎干细胞具有巨大的再生医学潜力，因为它们具有无限自我更新的能力，同时保持分化为所有成体细胞类型的能力。这项研究将增强我们对胚胎发育过程中干细胞沿着神经谱系定向分化的分子机制的理解。这些知识将为开发利用干细胞治疗神经退行性疾病和脑癌的强大策略提供基础。

DESCRIPTION (provided by applicant): Emerging evidence supports a central role for chromatin insulators in genome-wide organization of higher- order chromosome loop structures. The overall objective of this proposal is to examine the consequences of insulator-mediated chromatin organization during mouse embryonic stem (ES) cell differentiation and development. Our central hypothesis is that the vertebrate insulator CTCF partitions the genome into lineage-specific domains of co-expressed genes by facilitating the formation of higher-order chromatin loop structures in response to developmental cues. This hypothesis will be tested according to three specific aims. In Aim 1, live cell confocal microscopy will be leveraged to investigate CTCF distribution and dynamics in real time during ES cell commitment along the neural lineage. In Aim 2, genome-wide CTCF binding sites will be identified in ES cells using chromatin immunoprecipitation in combination with high- throughput, next generation sequencing (ChlP-Seq). Global CTCF occupancy maps for pluripotent ES cells will be compared to multipotent neuroprogenitors and terminally-differentiated neurons. Finally, in Aim 3, the structural organization of CTCF-based chromatin loops will be characterized using Chromosome- Conformation-Capture (3C) at genomic loci displaying differential insulator occupancy during neural lineage commitment. Correlation of these CTCF-based structures with recent genome-wide analyses of 'traditional' epigenetic modifications and lineage-specific gene expression profiles will provide a more global understanding of how the genome and the epigenome act in concert to regulate the formation of a diverse array of tissue-types during development. Completion of the proposed work will provide significant insight into the mechanisms that govern the commitment of pluripotent stem cells toward neuroectodermal lineages. This knowledge will enable advances in understanding the causes and consequences of higher- order chromatin structure during the onset of cancer and neurodegenerative diseases. Public Health Relevance: Embryonic stem cells have enormous potential for regenerative medicine due to their capacity for indefinite self-renewal while remaining poised for differentiation into all adult cell-types. The proposed research will enhance our understanding of the molecular mechanisms that regulate commitment of stem cells along the neural lineage during embryonic development. This knowledge will provide a foundation for the development of robust strategies which harness the therapeutic potential of stem cells for treatment of neurodegenerative diseases and brain cancer.