Study: Gene circuits reshape DNA folding and affect how genes are expressed

When a gene is turned on in a cell, it creates a ripple effect along the DNA strand, changing the physical structure of the strand. A new study by MIT researchers shows that these ripples can stimulate or suppress neighboring genes.These effects, which result from the winding or unwinding of neighboring DNA, are determined by the order of genes along a strand of DNA. Genes upstream of the active gene are usually turned up, while those downstream are inhibited.The new findings offer guidance that could make it easier to control the output of synthetic gene circuits. By altering the relative ordering and arrangement of genes, or “gene syntax,” researchers could create circuits that synergize to maximize their output, or that alternate the output of two different genes.“This is really exciting because we can coordinate gene expression in ways that just weren’t possible before,” says Katie Galloway, an assistant professor of chemical engineering at MIT. “Syntax will be really useful for dynamic circuits. Now we have the ability to select not only the biochemistry of circuits, but also the physical design to support dynamics.”Galloway is the senior author of the study, which appears today in Science. MIT postdoc Christopher Johnstone PhD ’26 is the paper’s lead author. Other authors include MIT graduate student Kasey Love, members of the lab of Brandon DeKosky, an MIT associate professor of chemical engineering, and researchers from Peter Zandsta’s lab at the University of British Columbia and the labs of Christine Mummery and Richard Davis at Leiden University Medical Center in the Netherlands.Gene syntaxWhen a gene is copied into messenger RNA, or “transcribed,” the double-stranded DNA helix must be unwound so that an enzyme called RNA polymerase can access the DNA and start copying it. That unwinding leads to physical changes in the structure of DNA strand.Upstream of the gene, DNA becomes looser, while downstream, it becomes more tightly wound. These changes affect RNA polymerase’s ability to access the DNA: Upstream of an active gene, it’s easier for the enzyme to attach; downstream, it’s more difficult.In a study published in 2022, Galloway and Johnstone performed computational modeling that explored how these biophysical changes might influence gene expression. They studied three different arrangements, or types of syntax: tandem, divergent, and convergent.Most synthetic gene circuits are designed in a tandem arrangement, with one gene followed by another downstream. In a divergent arrangement, neighboring genes are transcribed in opposite directions (away from each other), and in convergent syntax, they are transcribed toward each other.The modeling suggested that the divergent arrangement was most likely to produce circuits where both genes are expressed at a high level. Tandem arrangements were predicted to result in the downstream gene being suppressed by the upstream gene.In the new study, the researchers wanted to see if they could observe these predicted phenomena in human cells.“Normally, we think about gene circuits and pieces of DNA as these lines that we draw, but they’re polymers that have physical characteristics,” Galloway says. “The thing that we were trying to solve in this paper was: When you put two genes on the same piece of DNA, how does their physical interaction become coupled?”The researchers engineered circuits that each contained two genes, in either a tandem, divergent, or convergent configuration, into human cell lines and human induced pluripotent stem cells.The results confirmed what their modeling had predicted: In divergent circuits, expression of both genes was amplified. In tandem circuits, turning on the upstream gene suppressed the expression of the downstream gene.These effects produced as much as a 25-fold increase or decrease in gene expression, and they could be seen at distances of up to 2,000 base pairs between genes.Using a high-resolution genome mapping technique called Region Capture Micro-C, the researchers were also able to analyze how the DNA structure changed when nearby genes were being transcribed.As predicted, they found that the DNA regions downstream from an active gene formed tightly twisted structures known as plectonemes, similar to the tangles seen in a twisted telephone cord. These structures make it harder for RNA polymerase to bind to DNA.To engineer these cells, the researchers used a new system they developed with the LUMC team called STRAIGHT-IN Dual, which allows them to efficiently insert two genes into the same DNA strand at both alleles. This system is being reported in a second paper published today, in Nature Biomedical Engineering.Precise controlThe new findings could help guide the design of synthetic gene circuits, which are usually designed to be controlled by biochemical interactions with activator or repressor molecules. Now, circuit designers can also perform biophysical manipulations to enhance or repress genes expression.“Everyone thinks about the components they need, and the biochemical properties they need to build a circuit,” Galloway says. “Now, we have added the physical construction of those components, which is going to change how those biochemical units are interpreted.”As a demonstration of one potential application, the researchers built synthetic circuits containing the genes for two segments of a novel antibody discovered by the Dekosky lab, used to treat yellow fever, and incorporated them into human cells. As they expected, the divergent syntax produced larger quantities of the yellow fever antibody.Galloway’s lab has also used this approach to optimize the output of synthetic gene circuits they previously reported that could be used to deliver gene therapy or to reprogram adult cells into other cell types.This strategy could also be used to build a variety of other types of dynamic synthetic circuits, such as toggle switches, oscillators, or pulse generators, for any application that requires precise control over gene expression.“If you want coordinated expression, a divergent circuit is great. If you want something that’s either/or, you can imagine using a convergent or tandem circuit, so when one turns on, the other turns off, and you can alternate pulses,” Galloway says. “Now that we understand the syntax, I think this will pave the way for us to program dynamic behaviors.”The research was funded, in part, by the National Institutes of Health, the National Institute for General Medical Sciences, a National Science Foundation CAREER Award, the Pershing Square Foundation, the Air Force Research Laboratory, and the Koch Institute Support (core) Grant from the National Cancer Institute.