We often think of DNA as the classic double-helix structure we learned about in high school, consisting of two twisting chains made up of various sequences of four nucleotides: adenine, thymine, guanine, and cytosine. But DNA is hardly static, and it is not always arranged in the same double-helix structure. Rather, that form is constantly changing as DNA twists and unravels to enable transcription of mRNA from DNA.
As multiple polymerases (enzymes that bring about the synthesis of new RNA or DNA) dock on a fragment of DNA, natural coiling from double-stranded DNA is displaced to allow the DNA to separate locally into single strands, a necessary step of transcription. This makes hardly any difference in a single gene, but when polymerases cause multiple genes to be transcribed in the same or opposing directions, the dynamics of those coils can give rise to interference patterns. Sometimes, the twist in DNA can concentrate in certain regions, a phenomenon known as supercoiling. A build-up of supercoiling causes the entire molecule to begin rotating in space, a process known as DNA writhing.
Thus, the signature spiral form of DNA can serve multiple functions, and Enoch Yeung, an assistant professor in the UC Santa Barbara Mechanical Engineering Department, recently received a $640,000 National Science Foundation (NSF) Early CAREER Award to study the dynamics of DNA structure and how it affects genetic programs in living cells.
“We know that DNA can compact, because any chromosome is much longer than the cell that contains it, so it has to be compact to fit,” he notes. “The question is, how does it twist and writhe back on itself to achieve compactness? If you were to coil DNA randomly and hope that it fit somewhere, you would need a cell that was 5233 microns in size. That would be a big cell. But in bacteria, because of supercoiling, we can actually compact four or five million base pairs of DNA into less than one micron. That is an impressive process of compacting, one which we hope to understand and harness in the fields of synthetic and systems biology.”
Yeung’s CAREER Award proposal grew out of his desire to further the research he did as a PhD student at the California Institute of Technology, where he and colleagues showed that DNA supercoiling produced a new form of control between neighboring genes. They demonstrated that supercoiling gave rise to complex, previously overlooked patterns of gene expressions in the gene networks, patterns that are highly dependent on spatial variables, and that ended with relaxation of the supercoils. Yeung and his collaborators developed the first quantitative, multi-gene model to represent those dynamics. “The arrangement of neighboring genes around a gene impacts its transcription,” he explained. “No gene is an island in the genome.”
“We know that dynamic twisting is happening inside any linear fragment of the chromosome as part of an evolving landscape of transcription, but visualizing localized DNA supercoiling and writhing at a specific location of the genome inside a living cell has remained a challenge.” Yeung says. “We want to develop tools to do that in a simulated, test-tube setting, and, eventually, in vivo.” The NSF funding will enable him to develop cell-free and cell-based biotechnology to achieve that long-term goal.
The challenge begins, he says, “when we consider that naturally occurring genes are often part of a larger cluster of genes.” Any section of the genome can have a directionally aligned collection of genes transcribing toward each other or away from each other, with asymmetry in how many base pairs are transcribed in one direction or the other. We want to know how this spatial context affects DNA twisting as transcription of each of these genes occurs. Do specific conditions or specific arrangements favor expression of certain genes over others? Are there statistics that we can quantify as a function of these arrangements? Biophysically, can certain patterns of genetic arrangement cause excessive DNA writhing, to effectively shut off gene transcription in neighboring areas of the genome?”
Yeung has outlined three objectives for the CAREER project. The first is to develop new in vitro methods for measuring localized twisting and writhing in linear and circularized DNA, to better understand how local supercoiling density can alter or control nearby gene transcription. The second is to develop new in vivo biosensors that can measure the degree of localized twisting and writhing in DNA in living cells, as a way to link in vivo supercoiling to nearby transcriptional activity. Finally, he intends to develop novel biophysical models to describe how the spatial distribution and transport of localized supercoiling controls transcription in DNA.
“My long-term goals are to understand how DNA and cell fate are determined by DNA structure and to map the 3D global structure of the genome as a function of the localized processes of twisting and writhing,” he says. “In this project, we’ll be building synthetic DNA constructs to pursue the goal of visualizing and modeling all of the various dynamics of DNA in the presence of transcription.”
Various applications of this fundamental scientific work could eventually result, for instance, it might inform efforts to understand how bacterial pathogens become virulent, reflecting the previous finding that certain genes, many of which are related to virulence in salmonella and E. coli, are highly sensitive to the twisting state. Some are positively regulated by supercoiling, and others are negatively regulated by it.
To this point, Yeung says, “I’ve done experiments for the single scenario, where we’re looking at just the transcription by itself, and we have also looked at the scenarios with DNA writhing. We’d like to be able to visualize both at the same time and then build quantitative models that map the relationship between those two scenarios.”
The NSF grant includes funding for K-12 educational components, something Yeung plans to pursue via several hands-on educational and outreach activities for students at Santa Ynez High School and Lompoc High School. The goal of the program is to enable STEM-oriented high school students who may be economically disadvantaged to spend time at UCSB learning first-hand about scientific research and working with world-class equipment in biology workshops that allow students to design their own genetic experiments.
“If possible, we want to provide students with earlier exposure to alternative career paths focused in STEM, and help them see that DNA is more than just a picture in a textbook,” Yeung says. “It’s wonderful when you can find that talented student who has a natural curiosity about genetics and DNA but who might not otherwise have access to STEM experiences during their formative years.”