CAREER: Understanding and engineering DNA supercoiling-mediated feedback in gene circuits

合成的基因电路可以用来对细胞进行重新编程，以用于医学和生物技术的各种应用。这个项目的目标是确定DNA结构的变化如何影响合成基因回路的表达和一般的基因表达。该项目的结果将对基因和细胞治疗等应用的基因电路的设计产生广泛的影响。整合到这项研究中，有一个多样化的教育倡议计划，将参与、激励和培训下一代哺乳动物合成生物学家。虽然合成电路的逻辑设计已经投入了大量的努力，但关于遗传元件的新出现的三维结构对电路行为的影响却知之甚少。合成的基因回路可以作为模型微基因组系统来探索基因调控的生物物理基础。作为基因网络的简化模型，合成电路为研究转录网络中结构、功能和生物物理反馈之间的因果关系提供了一种工具。合成生物学旨在将合成基因控制系统整合到本地控制网络中，以指导细胞行为。然而，有限的能力，规定转基因表达和启动变化的天然基因表达限制了哺乳动物细胞的遗传编程。特别是，转录的随机性质使得跨多个遗传元件的协调表达具有挑战性。转录诱导了一波DNA超螺旋，改变了RNA聚合酶的结合亲和力，重塑了基因调控的生化格局。随着超螺旋的迅速扩散，转录动态地重塑了近端基因的调控，形成了一个复杂的反馈回路。通过利用生物物理调节，研究人员已经确定了一种偏向转录的随机性质并改变种群行为的机制。这项职业将使用合成基因电路来检验DNA超螺旋介导的反馈对转录的偶联和调节的普遍性。通过改变启动子的同一性、基因长度、基因间的间距和语法，这个项目将探索这些特征中的每一个如何影响表达谱。表达谱将通过结合使用显微镜和基于流式细胞术的技术在单个细胞中的RNA和蛋白质水平来测量。为了将基因活性和句法直接与结构联系起来，该项目将使用一系列基因组工具以高分辨率绘制DNA结构图，这些工具将在句法、转录活性和局部基因组结构之间建立因果关系。了解超级螺旋介导的反馈的设计原理将使研究人员能够定义一种基本的、被忽视的机制，细胞通过该机制协调复杂的任务。有了这样的理解，这项研究将重塑我们对基因调控的看法，并为阐明基因组中可能被疾病利用的机械脆弱点提供新的工具。这项工作将彻底改变我们对基因组调控的理解，同时建立一种全新的、正交的基因表达控制模式，可以利用这种模式来设计新功能、定制现有电路并实现强大的细胞工程。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Synthetic gene circuits can be used to reprogram cells for a variety of applications in medicine and biotechnology. The goal of this project is to determine how changes in DNA structure impact expression of synthetic gene circuits and gene expression in general. The results of this project will have a broad impact on the design of gene circuits for applications such as gene and cell therapies. Integrated into this research, there is a plan for diverse educational initiatives that will engage, inspire, and train the next generation of mammalian synthetic biologists. While significant efforts have been devoted to the logical design of synthetic circuitry, far less is understood regarding the impact of the emergent three-dimensional structure of genetic elements on circuit behavior. Synthetic gene circuits may serve as model microgenomic systems to explore the biophysical basis of gene regulation. As simplified models of gene networks, synthetic circuits provide a tool for examining the causal relationship between structure, function, and biophysical feedback in transcriptional networks. Synthetic biology aims to integrate synthetic genetic control systems within native control networks to direct cell behaviors. However, limited ability to prescribe transgene expression and to actuate changes in native gene expression constrains genetic programming of mammalian cells. In particular, the stochastic nature of transcription makes coordinating expression across multiple genetic elements challenging. Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. By harnessing biophysical regulation, the researchers have identified a mechanism for biasing the stochastic nature of transcription and changing the population behavior. This CAREER will use synthetic gene circuits to examine the generality of DNA supercoiling-mediated feedback to couple and tune transcription. By varying promoter identity, gene length, inter-gene spacing and syntax, this project will explore how each of these features impact expression profiles. Profiles of expression will be measured by RNA and protein levels in single cells using a combination of microscopy and flow cytometry-based techniques. To connect gene activity and syntax directly to structure, this project will map DNA structure at high resolution using a combination of genomic tools that will build a causal connection between syntax, transcriptional activity, and local genome structure. Understanding the design principles of supercoiling-mediated feedback will allow the researchers to define an essential and overlooked mechanism by which cells orchestrate complex tasks. With this understanding, the research will reshape our perspective on gene regulation and provide novel tools for illuminating mechanically fragile points within the genome that may be exploited by disease. This work will revolutionize our understanding of genome regulation while establishing an entirely new and orthogonal mode of gene expression control that can be harnessed to engineer novel functions, tailor existing circuits, and enable robust cell engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.