Genome organization of post-mitotic neurons in maturation and disease

项目总结/摘要 染色质的特征在于接近无膜核区室，如核纤层， 与抑制性异染色质或斑点有关，促进mRNA剪接。许多 跨越数十至数百个核糖核酸酶的基因组区域与这些核区室相互作用。这个水平 基因组组织的调控，并提供了一个三维（3D）的转录架构， 调控在有丝分裂后的成熟神经元中，与核室相关的基因组组织并不 已知的和新出现的证据表明，核隔间在很宽的范围内受到严重扰动， 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 组织与切割和运行技术（GO-CaRT）的开发，以确定染色质结构域， 与特定核区室-核纤层（纤层相关结构域，LAD）的接近，以及 核斑点（斑点相关域，SPAD）-在从小鼠和人脑分离的细胞中。在 人类神经前体细胞，SPAD富集了参与微管运动的基因， 这表明斑点调节着正在进行的关键细胞过程。在其他初步研究中， 发现高度富集已知的AD风险基因座。Tau病是一种进行性神经退行性疾病 由脑中不溶性tau聚集体定义。多项研究表明，关键斑点蛋白 变得错误定位于致病性tau的聚集体，严重破坏核斑点的结构。 基于这一科学背景和初步研究，这一建议的中心假设是， 在tau蛋白病中，斑点蛋白与tau聚集体的错误定位导致关键蛋白的斑点缔合的丢失。 神经元基因，这导致它们的转录和剪接缺陷。为了验证这一假设，目标1将 阐明基因组组织的成熟，在健康的，有丝分裂后的皮质神经元在小鼠中使用 GO-CaRT和相关的表观基因组学和转录组学方法。Aim 2将研究tau的影响 聚集体的基因组结构，使用建立的动物模型的致病性tau蛋白表达。 这些目标的成功实现将阐明基因调控与核转录的关系。 区室相关的基因组组织在体内神经元。结果还将提供有关如何 tau蛋白病中核区室的破坏可导致异常基因表达和剪接， 潜在地产生tau蛋白病如AD的发病机理的新模型。从训练 从这个角度来看，这个建议赞扬了申请人过去在生物信息学/计算方面的经验。 生物学与培训“湿实验室”分子生物学和人类神经疾病的动物模型。 在实验生物学的这些方面提供培训将是非常宝贵的申请人的职业兴趣 神经病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.