Spatiotemporal Dynamics of the Genome by 3D Orbital Tracking

通过 3D 轨道跟踪研究基因组时空动态

• 批准号: 10514845
• 负责人: Matthew Lee Ferguson
• 金额: $ 39.2万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10514845
• 关键词: 3-Dimensional; Address; Alternative Splicing; Bayesian Analysis; Binding; Biological Process; COVID-19; COVID-19 treatment; Cardiomyopathies; Cell Nucleus; Cells; Clinical; Collaborations; Complex; Computer Models; Corticosterone; Coupling; DNA; Data; Dexamethasone; Disease; Distal; Elements; Estrogen Receptors; Eukaryotic Cell; Event; Fluorescence; Fluorescence Microscopy; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Glucocorticoid Receptor; Goals; Hairy Cell Leukemia; Health; Hormones; Human; In Vitro; Institutes; Introns; Kinetics; Label; Laboratories; Length; Ligands; Light; Location; Malignant Neoplasms; Measurement; Measures; Methodology; Methods; Modeling; Molecular; Monitor; Myopathy; National Cancer Institute; Netherlands; Nuclear Export; Outcome; Parkinson Disease; Patients; Pharmacologic Substance; Positioning Attribute; Process; Proteins; RNA; RNA Processing; RNA Splicing; Raloxifene; Regulation; Regulatory Element; Reporter Genes; Research; Research Personnel; Response Elements; Sampling; Severe Acute Respiratory Syndrome; Side; Spectrum Analysis; Spliceosomes; Tamoxifen; Testing; Time; Transcript; Transcription Coactivator; Transcription Initiation; Transcriptional Activation; Universities; Work; acute myeloid leukemia cell; autism spectrum disorder; base; beta Globin; cell type; clinically relevant; experimental study; fluorescence imaging; genetic information; glucocorticoid-induced orphan receptor; in vivo; insight; malignant breast neoplasm; nervous system disorder; novel; physical model; public health relevance; receptor binding; response; single molecule; spatiotemporal; temporal measurement; time use; tool; transcription factor

The expression of genetic information depends on the fate of RNA transcripts. In Eukaryotic cells, this fate is determined by the successful execution of several processes, including transcription (initiation, elongation, and release) splicing, nuclear export and degradation. These processes are not independent in space or time. The rate and completion of any one process may influence that of another, and kinetic studies conducted on isolated components can be misleading or incomplete. Unfortunately, the coordinated kinetics of RNA processing events remains poorly understood primarily due to a lack of experiments that can either reproduce it in vitro or visualize it in vivo. The main goal of our research is to understand the molecular mechanisms underlying the functioning of the living genome. Our main tool is live cell single molecule fluorescence microscopy. By fluorescently labeling protein and RNA molecules and DNA elements within living cells we can determine the spatiotemporal relationships between them that govern gene expression and RNA splicing at an active gene in a living cell. Our most recent work has measured the timing of gene activation by the transcription factor Glucocorticoid receptor after binding to dexamethasone and the temporal coordination of transcription[1,2] and splicing of intron 2 of a human beta globin reporter gene[3]. We do this using novel single molecule approaches and cutting edge live cell single molecule fluorescence microscopy. Our methods are based on fluorescence correlation spectroscopy which utilizes high temporal resolution to characterize changes in fluorescence intensity at a location in space and time and relates that to molecular concentrations, interactions and dwelltime using physical and computational models. Models are tested using Bayesian inference criterion. This research will directly benefit patients suffering from SARS, Breast cancer, AML and hairy cell leukemia's by showing the molecular mechanism leading to disease and clinical outcomes. It will also open new research avenues into the molecular basis of neurological and muscle diseases with origins in alternative splicing mis regulation such as Parkinson's disease, Autism, ALS and Cardiomyopathies.

遗传信息的表达取决于RNA转录物的命运。在真核细胞中， 命运由几个过程的成功执行决定，包括转录（起始，延伸， 和释放）拼接、核输出和降解。这些过程在空间和时间上都不是独立的。 任何一个过程的速度和完成可能会影响另一个过程， 孤立的组件可能会误导或不完整。不幸的是，RNA加工的协调动力学 事件仍然知之甚少，主要是由于缺乏可以在体外重现它的实验， 在活体中进行可视化。 我们研究的主要目标是了解这些功能的分子机制。 活基因组。我们的主要工具是活细胞单分子荧光显微镜。通过荧光标记 蛋白质、RNA分子和DNA元件，我们可以确定它们的时空位置。 它们之间的关系决定了活细胞中活性基因的基因表达和RNA剪接。我们 最近的工作测量了转录因子糖皮质激素受体对基因激活的时间 在与地塞米松结合以及转录[1，2]和剪接的时间协调后， 人β珠蛋白报告基因[3]。我们使用新颖的单分子方法和尖端的实时 细胞单分子荧光显微术。我们的方法是基于荧光相关光谱 其利用高时间分辨率来表征空间中某一位置处荧光强度的变化 并将其与分子浓度、相互作用和停留时间联系起来， 计算模型使用贝叶斯推理标准对模型进行检验。 这项研究将直接使SARS、乳腺癌、AML和毛细胞白血病患者受益 通过展示导致疾病和临床结果的分子机制来研究白血病。它还将开启新的 选择性剪接引起的神经和肌肉疾病的分子基础的研究途径 失调如帕金森氏病、自闭症、肌萎缩侧索硬化症和心肌病。