New targeted method to probe the function of 3D chromosomal structure

UNIVERSITY PARK, Pa. — A new method capable of inducing interactions between specifically chosen locations on the genome allows researchers to begin to identify the causal relationship between the three-dimensional structure of chromosomes and genome function. A paper by Penn State researchers describing the method, called “chemically induced chromosome interaction (CICI),” and two functional tests of the method appear Feb. 9 in the journal Nature Communications.

The genomes of eukaryotes — organisms ranging from yeast to humans whose cells have a distinct nucleus — are made up of chromosomes. Inside the nucleus, chromosomes, which are long, linear strands of DNA packed with many proteins that carry genetic information, are arranged in a three-dimensional conformation that, depending on the cell type, can bring genomic regions linearly distant from one another in close enough contact to interact functionally. These interactions are thought to be important for things like gene regulation, which controls when and where certain genes are used by the cell.

“It is now quite simple to sequence DNA and identify functional units like genes and regulatory regions, but understanding how the genome actually works is more complicated,” said Lu Bai, associate professor of biochemistry and biology. Molecular and Physics at Penn State and leader of the research team. “We know that the three-dimensional structure of the genome, including the interactions between different genomic regions on a chromosome or between two chromosomes, can be important for the function of the genome, so we wanted to develop a generalizable method to causally link this structure to the function. .”

The three-dimensional structure of the genome has been the subject of numerous recent studies using various “chromosomal conformation capture” techniques. Generally, these techniques work by chemically cross-linking the DNA and proteins of the chromosomes forming bonds that lock all areas where the chromosomes are close to each other in three-dimensional space and can interact. Cross-linked genomes can then be broken down, manipulated, and sequenced to identify regions of the genome that have been locked together.

“These methods have been extremely effective in identifying regions of the genome that are linearly distant on chromosomes but close to each other in three-dimensional space,” Bai said. “But we want to know if this chromosomal conformation is important. Comparisons of chromosomal conformation and functional measures, like gene expression, between different cell types can allow us to correlate structure with function, but do not directly test causation. Methods that can test causation directly that have been developed often have other problems, such as off-target impacts that more generally disrupt cellular function, making results more difficult to interpret.

The research team developed a method – CICI – to solve these problems. In short, they can insert short DNA sequences into any two locations in the genome, express artificially engineered proteins that bind to those inserted sequences, and then chemically induce the proteins to bind to each other when in close proximity. . They can then compare cellular functions before and after the induction of the structural change. Importantly, the system does not disrupt any normal cellular function beyond the regions tested.

“Essentially, we can put two sticky pads anywhere in the genome, and the genome behaves normally until we ‘turn on’ the sticky pads with a chemical signal,” Bai said. “So we can directly compare cell function before and after the change.”

The team tested their method in yeast cells and showed that the two selected regions did not interact before chemical induction and interacted strongly after induction. They also performed two functional experiments showing that three-dimensional structure is important for DNA repair in the yeast mating type switch system and that it does not appear to impact the timing of replication of DNA.

“Our method allows us to target specific locations in the genome and directly test the functional impact of artificially forcing regions to interact without any off-target effects,” Bai said. “We can also use this method to look at chromosome dynamics by following the two regions through the cell cycle to see how long the regions take to meet. Although we developed the method using yeast as a model system, there is no reason why it cannot be extended to mammalian cells. We hope to be able to demonstrate this in the future.

In addition to Bai, the Penn State research team includes Manyu Du, Fan Zou, Yi Li, and Yujie Yan. The research was supported by the US National Institutes of Health and the US National Science Foundation.

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