Genome organization of post-mitotic neurons in maturation and disease

项目摘要/摘要 染色质的特征可以是接近无膜的核隔室，例如板层， 它与抑制性异染色质或斑点有关，促进了mRNA的剪接。许多 跨越数十到数百个千碱基的基因组区域与这些核隔间相互作用。这一级别 基因组组织的结构是受调控的，并为转录 监管。有丝分裂后成熟神经元中的核室相关基因组组织不是 已知的，并且正在出现的证据表明，核隔间在广泛的 神经退行性疾病，包括阿尔茨海默病(AD)。最近，一项名为基因组的新技术 采用切割和运行技术的组织(GO-CART)被开发来识别染色质结构域 靠近特定的核隔室--核层(层相关区域，LAD)，以及 从小鼠和人脑分离的细胞中的核斑点(斑点相关区域，SPADs)。在……里面 人类神经前体细胞，SPAD富含参与微管运动的基因， 这表明斑点调控着正在进行的关键细胞过程。在其他初步研究中，SPAD是 已知的阿尔茨海默病风险基因高度丰富。肌萎缩侧索硬化症是一种进行性的神经退行性疾病 定义为大脑中不可溶的tau聚集体。多项研究表明，关键的斑点蛋白 错误地定位于致病tau的聚集体，严重破坏了核斑点的结构。 基于这一科学背景和初步研究，这一提议的中心假设是 Tauopathy中斑点蛋白与tau聚合体的错误定位导致Key的斑点关联丢失 神经元基因，导致其转录和剪接缺陷。为了检验这一假设，目标1将 在健康的小鼠皮质神经元中阐明基因组组织的成熟 Go-Cart以及相关的表观基因组学和转录组学方法。然后，目标2将调查tau的影响 聚集在基因组结构上，使用已建立的致病tau表达的动物模型。 这些目标的成功完成将阐明基因调控和核之间的关系 活体神经元中的间隔室相关基因组组织。结果还将提供关于如何 肌萎缩侧索硬化症中核室的破坏可能导致基因表达和剪接异常， 潜在地产生了新的阿尔茨海默病等疾病的发病机制模型。从一次培训中 从角度来看，这项建议是对申请人过去在生物信息学/计算方面的经验的赞扬 受训于“湿实验室”分子生物学和人类神经疾病动物模型的生物学。 提供这些方面的实验生物学培训对申请者的职业兴趣将是无价的 神经学和神经退行性疾病。

PROJECT SUMMARY/ABSTRACT Chromatin can be characterized by proximity to membraneless nuclear compartments such as the lamina, which is associated with repressive heterochromatin, or speckles, which facilitate mRNA splicing. Many genomic regions spanning tens to hundreds of kilobases interact with these nuclear compartments. This level of genome organization is regulated and provides a 3-dimensional (3D) architecture for transcriptional regulation. Nuclear compartment-associated genome organization in post-mitotic, mature neurons is not known, and emerging evidence indicate that nuclear compartments are grossly perturbed in a wide range of neurodegenerative diseases including Alzheimer’s disease (AD). Recently, a new technique called Genome Organization with Cut and Run Technology (GO-CaRT) was developed to identify chromatin domains in proximity to specific nuclear compartments – the nuclear lamina (lamina associated domains, LADs), and nuclear speckles (speckle associated domains, SPADs) – in cells isolated from mouse and human brain. In human neural precursor cells, SPADs are enriched for genes involved in microtubule-based movement, suggesting that speckles regulate ongoing key cellular processes. In other Preliminary Studies, SPADs were found to be highly enriched for known AD risk loci. Tauopathies are progressive, neurodegenerative diseases defined by insoluble tau aggregates in the brain. Multiple studies have demonstrated that key speckle proteins become mislocalized to aggregates of pathogenic tau, severely disrupting the structure of nuclear speckles. Based on this scientific background and Preliminary Studies, the central hypothesis of this proposal is that the mislocalization of speckle proteins to tau aggregates in tauopathies leads to loss of speckle-association of key neuronal genes, which results in defects in their transcription and splicing. To test this hypothesis, Aim 1 will elucidate the maturation of genome organization in healthy, post-mitotic cortical neurons in the mouse using GO-CaRT and related epigenomic and transcriptomic methods. Aim 2 will then investigate effect of tau aggregates on genome architecture, using established animal models of pathogenic tau expression. Successful completion of these Aims will elucidate the relationship between gene regulation and nuclear compartment-associated genome organization in neurons in vivo. Results will also provide insights into how disruption of nuclear compartments in tauopathies can contribute to aberrant gene expression and splicing, potentially generating new models of the pathogenesis of tauopathies such as AD. From a training perspective, this proposal compliments the applicant’s past experience with bioinformatics/computational biology with training in “wet lab” molecular biology and animal models of human neurological disease. Providing training in these aspects of experimental biology will be invaluable to the applicant’s career interests in neurology and neurodegenerative disorders.