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

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

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 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.