4D Movies Reveal Two Mechanisms For DNA Loops

By Allison Proffitt 

October 6, 2017 | Researchers from Baylor College of Medicine, Rice University, Stanford University and the Broad Institute of MIT and Harvard have created the first high-resolution 4D map of genome folding, tracking an entire human genome as it folds over time. The progressive images reveal details about two different mechanisms of DNA folding within the nucleus, and how the folding impacts genome function.

The work was published yesterday in Cell (DOI: 10.1016/j.cell.2017.09.026).

“There’s a fine-tuned balance between these two phenomena that are both rapidly creating these very specific contacts in the genome, even though the nucleus is such a crowded place,” Suhas Rao, first author on the paper, told Bio-IT World.

The team is one of several exploring how DNA folds into the nucleus and its implications. In 2014, the same team used the in situ Hi-C protocol to comprehensively map chromatin contacts genome-wide (the work was also published in Cell, DOI: 10.1016/j.cell.2014.11.021). The Hi-C protocol combines DNA proximity ligation with high-throughput sequencing in a genome-wide fashion. “The Hi-C protocol works by gluing the genome together, cutting it into millions of pieces, and taking these blobs that have pieces that came into close proximity but didn’t necessarily come from the same place in the [linear] genome,” Rao explained in 2014.

The new study takes that map and sets it in motion.

“Previously we developed an improved method of the Hi-C protocol to make really high resolution maps of DNA interactions; we used that protocol here,” Rao said. “The key distinction is, instead of doing it once and taking a snapshot of the interactions, we do it over a time course and we actually make a movie.”

The movie has a “modest frame rate”, Rao joked, but it does allow researchers to watch DNA loops move and change over time, revealing hints of what may be driving the arrangements and how the timing proceeds.

To make changes, the research team began by disrupting cohesin, a ring-shaped protein complex located at the boundaries of nearly all known loops. In a colon cancer cell line, the team was able to quickly remove and then add the cohesin complex, and watch the genome respond.

For some types of loops and loop domains, the genome responded as they expected. Within minutes of removing cohesin, thousands of loops disappeared. When cohesin was replaced, the loops quickly formed again—some within 30-40 minutes, others over the course of a few hours.

But all the genome structure didn’t respond to removing cohesin. “This one class of loops—which forms by extrusion, depends on cohesin, and pinches together two pieces of the genome—they’re all lost as soon as you get rid of cohesin,” Rao explained. “On the other hand, you have this class of loops, where it brings together elements across the genome, across chromosomes, and once you get rid of cohesin, these loops get stronger.” The team calls the second mechanism compartmentalization.

The second class of loops, Rao and his colleagues observed, forms between super enhancers, areas of the genome that control key developmental and disease-associated genes. “We see that these… super enhancers all co-localize as soon as we get rid of cohesin,” he said. “We were really excited to see this phenomenon happening where, basically, these super enhancers form loops with each other via compartmentalization, not by extrusion.”

The two loop-forming mechanisms seem to interfere with each other. When cohesin is present, loops formed by extrusion keep super enhancers interacting with nearby genes, “keeping them in their local neighborhood,” Rao explains. “When cohesin is gone, super enhancers have a tendency to segregate amongst themselves in the nucleus… Genes that are nearby get mis-regulated and down-regulated. Basically the gene express program of the cell gets affected.”

The findings suggest many new questions, and Rao rattled off plenty of areas for work: How many cohesin complexes are there? What is the motor process that is driving this? Is it cohesin by itself? Are there other motor proteins?

Cohesin is known to play a role in several developmental diseases and cancers, and for many cohesin-associated proteins are known to be mutated. “Over the last decade or so, these diseases—these cohesinopathies—have been identified. But the mechanisms of pathogenicity is still very unclear. It’s been pursued from a number of different angles. Our thinking is that the genome-folding angle is crucial to understanding the pathogenicity of these diseases,” Rao said.

That, of course, suggests the biggest questions: “How can we take this known mutation and figure out what’s actually going wrong mechanistically. How is that causing the disease? It’s still very early days, but there are a lot of exciting directions.”