Identification of the active nuclear niche(s) using novel proteomic, genomic, transgenic, and live-cell microscopy technologies

基因表达的研究和缩合物在调节基因表达中的可能作用， 基本上忽略了已知的核结构。这个建议是重要的，因为我们提出了一个新的模型， 核组织在基因表达调控中作用：1）核斑点和尚不清楚的核 隔室/体帮助组织其他相分离的缩合物以调节基因表达; 2） 核散斑与周围的核隔室/核体以及相分离的相关 凝聚物一起代表可能具有不同功能性质的活性核龛; 3）小 距离很重要：抑制性基因和这些不同的活性基因之间只有几百nm的基因运动， 核小生境可能差异调节基因表达; 4）在一个距离上的差异：成分流入和 5）这些相同的核区室将对基因表达产生全局影响; 区室/体可以类似地调节RNA加工并组织核输出。 在这里，我们建议：1）确定已知和仍然未知的核"活性"的多个组件 2）绘制全基因组范围内基因相对于这些活跃小生境的位置和预测运动 3）可视化核体/隔室动力学和蛋白质通量 核体之间的稳定状态，并通过生理过渡; 4）可视化的运动， 报告转基因，内源基因，以及与这些核基因相关的重连染色体基因座， 在时间上将基因表达的变化与它们的动态运动相关联， 5）在RNA合成过程中观察前体mRNA和核mRNA的运动 处理和输出; 6）测量核体成分到和来自相邻转录的通量 染色质此外，我们建议开发相对低成本，新颖的显微镜平台和软件 专门设计用于在我们的实验室以及其他实验室中实现这些活细胞成像目标。 我们的目标是：1。使用蛋白质、基因、RNA相对于活性核区室的图谱 TSA-MS-Ratio、通过光学显微镜验证和TSA-Seq的迭代轮次; 2.测量动态 体，使用活细胞成像的核体的组件; 3.测量时间相关性， 相对于核体的基因表达和基因运动的变化，并可视化 表达成绩单; 4.设计并交付两款新型显微镜，以便于实现目标1 - 3， 价格适中。这些目标的成功实现将大大改变我们目前对 核组织在调节基因表达中的作用，对广泛的研究领域产生影响。

The study of gene expression and possible role of condensates in regulating gene expression have largely ignored known nuclear structures. This proposal is significant because we propose a novel model for the role of nuclear organization in regulating gene expression: 1) Nuclear speckles and still unknown nuclear compartments/bodies help organize other phase-separated condensates to modulate gene expression; 2) Nuclear speckles together with surrounding nuclear compartments/bodies and associated phase-separated condensates together represent active nuclear niches which may have different functional properties; 3) Small distances matter: gene movements of only a few hundred nm between repressive and these different active nuclear niches may differentially regulate gene expression; 4) Action-at-a distance: component flux into and out of these nuclear compartments will have global effects on gene expression; 5) These same nuclear compartments/bodies may similarly modulate RNA processing and organize nuclear export. Here we propose to: 1) Identify multiple components of known and still unknown nuclear “active niches”; 2) Map genome-wide the positions and predicted movements of genes relative to these active niches during physiological transitions; 3) Visualize nuclear body/compartment dynamics and fluxes of proteins between nuclear bodies in steady-state and through physiological transitions; 4) Visualize movements of reporter transgenes, endogenous genes, and rewired chromosome loci relative to these nuclear bodies/compartments and temporally correlate changes in gene expression with their dynamic movements and compartment associations; 5) Visualize movements of pre-mRNAs and nuclear mRNAs during RNA processing and export; 6) Measure fluxes of nuclear body components to and from adjacent transcribing chromatin. Additionally, we propose developing relatively low-cost, novel microscope platforms and software specifically designed to facilitate these live-cell imaging goals in our laboratories as well as others. Our Aims will be to: 1. Map proteins, genes, RNAs relative to active nuclear compartment(s) using iterative rounds of TSA-MS-Ratio, validation by light microscopy, and TSA-Seq; 2. Measure dynamics of bodies, components of nuclear bodies using live-cell imaging; 3. Measure temporal correlation between changes in gene expression and gene movement relative to nuclear bodies and visualize the export path of expressed transcripts; 4. Design and deliver two novel microscopes designed to facilitate Aims 1-3 at a modest cost. Successful completion of these Aims should significantly change our current understanding of the role of nuclear organization in regulating gene expression with impact across a wide range of research fields.