Biophysical modeling of cis-regulatory complexes in transcription and splicing using massively parallel reporter assays

使用大规模并行报告分析对转录和剪接中的顺式调控复合物进行生物物理建模

• 批准号: 10472049
• 负责人: JUSTIN B. KINNEY
• 金额: $ 48万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472049
• 关键词: Antisense Oligonucleotides; Bacteria; Basic Science; Biological; Biological Assay; Biological Sciences; Biophysics; Bypass; Cells; Complex; DNA; Data; Defect; Environment; Escherichia coli; Exons; Gene Expression; Genetic Transcription; High-Throughput DNA Sequencing; Human; Mathematics; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Computers; Molecular Machines; Nucleic Acids; Organism; Pathogenicity; Physics; Proteins; RNA; RNA Splicing; Reporter; Research; Site; System; Therapeutic; Training; Transcriptional Regulation; Work; biological systems; biophysical model; design; direct application; experience; experimental study; genetic regulatory protein; genetic variant; human disease; improved; in vivo; innovation; mathematical model; novel strategies; programs; promoter; protein protein interaction; response; synthetic biology

PROJECT SUMMARY / ABSTRACT Gene expression in all organisms is controlled by large protein-nucleic-acid assemblies called “cis-regulatory complexes.” From transcription in bacteria to mRNA splicing in humans, cis-regulatory complexes act as molecular computers, tuning gene expression in response to information in the cellular environment. A mechanistic understanding of how these complexes function will have a major impact on basic science, synthetic biology, and human disease. This level of understanding requires biophysical models that quantitatively account for the protein-DNA, protein-RNA, and protein-protein interactions that occur within each cis-regulatory complex. Such models have been established for a handful of intensively studied systems, such as the lac promoter of Escherichia coli. However, the experiments used to establish these models require quantitative control over the in vivo concentrations of regulatory proteins, a requirement that is very hard to meet in less-well- understood contexts. In the coming years, my lab will pursue an alternative approach to deciphering biophysical models of cis-regulatory complexes in living cells. This innovative approach is highly scalable and applicable to a wide variety of biological systems. Our experiments will leverage massively parallel reporter assays performed on synthetic regulatory sequences that are designed to probe specific macromolecular interactions. These data will be used to decipher expression manifolds, mathematical objects whose inference bypasses the need to experimentally control in vivo protein concentrations. This program thus combines my training in theoretical physics and my extensive experience using high-throughput DNA sequencing to measure biophysical quantities. To emphasize the full generality of this approach, I am proposing work in two diverse biological contexts: transcriptional regulation in E. coli (Project 1) and alternative mRNA splicing in human cells (Project 2). Project 1a will establish the capabilities and limitations of this approach in a well-understood bacterial system, while Project 1b will extend this approach to bacterial promoters about which little is yet known. Project 2a will develop a biophysical model for the integration of information encoded within 5ʹ and 3ʹ splice sites during exon definition. Project 2b will use biophysical modeling to better understand and guide improvements in antisense oligo treatments that correct splicing defects in human disease. Project 2 is not predicated on Project 1, but the strategies developed in our studies of bacterial transcription will inform and improve our studies of splicing in humans. This research program will thus establish a new approach for dissecting cis-regulatory complexes in a wide range of biological systems. It will also yield specific biophysical models that can be immediately and broadly applied to problems in synthetic biology, to the prediction of pathogenic genetic variants, and to the design of molecular therapeutics.

项目概要/摘要 所有生物体中的基因表达均由称为“顺式调节”的大型蛋白质核酸组装体控制 从细菌的转录到人类的 mRNA 剪接，顺式调控复合物发挥着作用 分子计算机，根据细胞环境中的信息调整基因表达。 对这些复合物如何发挥作用的机制理解将对基础科学、合成科学产生重大影响 这种水平的理解需要定量的生物物理模型。 解释每个顺式调控中发生的蛋白质-DNA、蛋白质-RNA 和蛋白质-蛋白质相互作用 已经为一些经过深入研究的系统建立了这样的模型，例如 lac。 然而，用于建立这些模型的实验需要定量。 控制调节蛋白的体内浓度，这一要求在不太好的情况下很难满足 在未来几年中，我的实验室将寻求一种替代方法来破译生物物理学。 这种创新方法具有高度可扩展性并适用于活细胞中的顺式调控复合物模型。 我们的实验将利用大规模并行报告分析。 对旨在探测特定大分子相互作用的合成调控序列进行。 这些数据将用于破译表达式流形，即其推理绕过的数学对象 因此，需要通过实验控制体内蛋白质浓度。 理论物理学和我使用高通量 DNA 测序测量生物物理的丰富经验 为了强调这种方法的全面普遍性，我建议在两个不同的生物学领域开展工作。 背景：大肠杆菌中的转录调控（项目 1）和人类细胞中的选择性 mRNA 剪接 （项目 2）。项目 1a 将在充分了解的细菌中建立这种方法的能力和局限性。 系统，而项目 1b 将把这种方法扩展到细菌启动子，而对此我们知之甚少。 2a 将开发一个生物物理模型，用于整合 5ʹ 和 3ʹ 剪接位点内编码的信息 项目 2b 将使用生物物理模型来更好地理解和指导改进。 纠正人类疾病剪接缺陷的反义寡核苷酸治疗并非以项目为基础。 1，但是我们在细菌转录研究中开发的策略将为我们的研究提供信息并改进 因此，该研究计划将建立一种剖析顺式调控的新方法。 它还将产生可用于广泛生物系统中的特定生物物理模型。 立即广泛应用于合成生物学问题，预测致病遗传变异， 以及分子疗法的设计。