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绝缘体介导的染色质组织在小鼠胚胎干细胞分化和发育过程中的后果。我们的中心假设是，脊椎动物的绝缘子CTCF通过促进高阶染色质环结构的形成来响应发育提示，将基因组划分为共同表达基因的谱系特异性区域。这一假设将根据三个具体目标进行检验。在目标1中，活细胞共聚焦显微镜将被用来实时研究在ES细胞沿神经谱系的定位过程中CTCF的分布和动态。在目标2中，将使用染色质免疫沉淀和高通量下一代测序(ChlP-Seq)相结合的方法在ES细胞中鉴定全基因组CTCF结合位点。多能胚胎干细胞的全球CTCF占位图将与多能神经前体细胞和终末分化的神经元进行比较。最后，在目标3中，将使用染色体构象捕获(3C)来表征基于CTCF的染色质环的结构组织，该基因座在神经谱系承诺期间显示不同的绝缘子占据。将这些基于CTCF的结构与最近全基因组范围内对传统的表观遗传修饰和特定谱系的基因表达谱的分析相关联，将提供对基因组和表观基因组如何协同作用以调节发育期间不同组织类型阵列的形成的更全面的理解。拟议工作的完成将为控制多能干细胞向神经外胚层谱系承诺的机制提供重要的洞察力。这一知识将使人们在理解癌症和神经退行性疾病发病过程中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，同时仍有可能分化为所有成体细胞类型。这项拟议的研究将加深我们对胚胎发育过程中干细胞沿神经谱系承诺的分子机制的理解。这些知识将为开发强有力的战略提供基础，这些战略利用干细胞的治疗潜力治疗神经退行性疾病和脑癌。

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.