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

项目总结/摘要 所有生物体中的基因表达都是由称为“顺式调控”的大的蛋白质-核酸组装体控制的。 情结”从细菌中的转录到人类中的mRNA剪接，顺式调节复合物充当 分子计算机，根据细胞环境中的信息调整基因表达。一 对这些复合物如何起作用的机械理解将对基础科学、合成化学和生物化学产生重大影响。 生物学和人类疾病。这种理解水平需要生物物理模型， 解释了蛋白质-DNA，蛋白质-RNA和蛋白质-蛋白质相互作用，这些相互作用发生在每个顺式调节蛋白质中。 复杂.这样的模型已经建立了一些深入研究的系统，如lac 大肠杆菌的启动子。然而，用于建立这些模型的实验需要定量的 控制调节蛋白的体内浓度，这是一个很难满足的要求， 理解上下文。在接下来的几年里，我的实验室将寻求另一种方法来破译生物物理学 活细胞中顺式调节复合物的模型。这种创新方法具有高度可扩展性，适用于 各种各样的生物系统。我们的实验将利用大规模平行的报告分析 在设计用于探测特定大分子相互作用的合成调控序列上进行。 这些数据将被用来破译表达式流形，数学对象的推理绕过 需要通过实验控制体内蛋白质浓度。因此，该计划结合了我的培训， 理论物理和我的丰富经验，使用高通量DNA测序来测量生物物理 是介于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：E.大肠杆菌（项目1）和人类细胞中的可变mRNA剪接 （项目2）。项目1a将在一个众所周知的细菌中建立这种方法的能力和局限性。 系统，而项目1b将这种方法扩展到细菌启动子，其中知之甚少。项目 2a将开发一个生物物理模型，用于整合在5 ′和3 ′剪接位点内编码的信息， 外显子定义项目2b将使用生物物理建模来更好地理解和指导改进， 反义寡核苷酸治疗，纠正人类疾病中的剪接缺陷。项目2不是基于项目 1，但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和改进， 在人体内进行剪接。因此，这项研究计划将建立一个新的方法，解剖顺式调节 复合物在广泛的生物系统中。它还将产生特定的生物物理模型， 立即并广泛地应用于合成生物学的问题，预测致病性遗传变异， 以及分子治疗的设计。

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.