Gene syntax defines supercoiling-mediated transcriptional feedback.

Gene syntax—the order and arrangement of genes and their regulatory elements—shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus perturbs the transcription of adjacent genes, but the molecular basis of this effect remains poorly understood. In this work, we show that supercoiling-mediated feedback arises from transcription and regulates expression of adjacent genes in a syntax-specific manner. Using a suite of assays, we measured syntax- and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter, we built and improved compact gene circuits, tuning the expression mean, noise, and stoichiometry across delivery methods and cell types. Integrating supercoiling mediated feedback into models of gene regulation will expand our understanding of native and synthetic systems. Editor's summary: Transcription reshapes DNA folding within living cells, driving patterns of gene expression. However, this process remains poorly understood, which limits the effectiveness of gene circuits for therapeutic applications. Johnstone et al. investigated how transcription generates DNA supercoiling (changes in DNA twist) and how these emergent structures tune the expression of adjacent genes. They found that altering gene syntax, defined by the relative order and orientation of neighboring genes, changed DNA supercoiling and affected both the level and variability of gene expression. This work helps explain patterns of natural gene regulation and may improve the design of gene therapies. —Di Jiang INTRODUCTION: Native gene networks require coordination of multiple transcriptional units to robustly pattern cell fate. Although precise control of gene expression is crucial for this process, the coupling between closely colocalized genes, including both native regulatory circuits and size-limited, synthetic gene circuits, is not fully understood. We aimed to uncover how transcription of a single gene affects expression of adjacent genes through the over- and undertwisting of DNA caused by RNA polymerase motion. This twisting of DNA is known as DNA supercoiling and arises from the directionality of transcription. Supercoiling impacts transcription of adjacent genes by altering RNA polymerase binding, forming a feedback loop. Supercoiling has been studied in silico, in vitro, and genome wide, but perturbative, locus-specific supercoiling measurements in living cells are more difficult, limiting mechanistic dissection of this phenomenon and its impact on gene expression. RATIONALE: We measured the impact of supercoiling on gene expression by building and integrating synthetic two-gene reporter circuits in mammalian cell lines. These synthetic circuits allowed us to probe how gene syntax, the relative order and orientation of genes, determines supercoiling-mediated feedback while holding gene identity, circuit copy number, genomic location, and other parameters constant. By inducing expression of one of the genes with a small molecule, we directly varied transcription-generated supercoiling. We measured single-cell protein and mRNA levels using flow cytometry, allowing us to calculate both the mean and variability of each reporter gene across a population of cells. Using circuits integrated at a fixed safe harbor locus, we quantified the local epigenetic state using CUT&Tag assays, the positive supercoiling density using GapRUN, and the chromatin folding behavior using Region Capture Micro-C. These measurements provided a high-resolution view of chromatin behavior at the circuit locus and the response to induction of the inducible gene. RESULTS: We found that gene syntax shapes expression profiles in a syntax- and induction-dependent manner. Pairs of genes with divergent syntax show amplified, correlated expression, whereas pairs with tandem syntax have reduced expression from the downstream gene. These syntax-dependent behaviors generalize well across both integration method and cell type. Expression patterns also co-occur with changes in chromatin state. Both direct measurements of positive DNA twist and indirect correlates of chromatin structure suggest that supercoiling contributes to these syntax-dependent behaviors. Applying our observations to engineer synthetic circuits, we improved antibody production yield, tuned stoichiometric expression ratios, and optimized the function of all-in-one inducible circuits by changing only gene syntax. CONCLUSION: Supercoiling-mediated feedback couples genes spaced within a few kilobases. This phenomenon may contribute to the coordination of native transcriptional programs. Applying the design rules suggested by our work can enhance the predictability, performance, and functional range of engineered gene circuits. Supercoiling-mediated feedback and gene syntax couple the expression of adjacent genes.: Supercoiling couples the motion of elongating RNA polymerases to the transcriptional initiation rate of adjacent genes. Influenced by this supercoiling-mediated feedback, the different gene syntaxes show distinct patterns of expression and chromatin states. Using supercoiling-informed design rules, engineered gene circuits can be optimized for robustness and performance. 3D, three dimensional. [ABSTRACT FROM AUTHOR]