Scientists at Fred Hutchinson Cancer Research Center have worked out the molecular underpinnings of how chromosomes make the right number of crossovers — important links that make it possible for developing sex cells (eggs or sperm in humans) to sort those chromosomes properly. Crossovers are a little like Goldilocks’ porridge — they need to be just right. Too few or too many crossovers, and new cells end up with the wrong number of chromosomes, which can cause miscarriages or developmental disorders.
It’s been known for 100 years that our chromosomes have a way of preventing too many crossovers along their length. What’s been missing all that time has been a working model that identifies the key molecules involved.
In work published today in the Proceedings of the National Academy of Sciences, Hutch molecular biologist Dr. Gerry Smith and his team outline just such a model in yeast that explains how chromosomes find their happy medium during sex-cell formation.
“What’s significant is that we’ve developed a molecular model of the proteins involved and how they work together to create crossover interference,” said Smith, the study’s senior author.
Creating a sperm or egg cell is an incredibly complex process. Among the many vital steps, genetic material packaged in chromosomes — half from mom and half from dad — must be faithfully copied and precisely parceled out to the new cells.
Crossovers are needed during the parceling process. They occur when sections of the maternal and paternal versions of chromosomes overlap and connect. These connections create tension that helps chromosomes properly pull apart as the cell divides, ensuring each new cell ends up with exactly the right set of genetic material.
So how do cells regulate this process to avoid too many, or too few, crossovers?
A phenomenon called crossover interference, in which a crossover at one location along a chromosome reduces the instances of another crossover nearby, was first observed in 1915, Smith said. It was discovered in fruit flies and then in most other organisms tested. But it wasn’t until 1990 that anyone proposed an idea of how it might work.
“There was no molecular model,” he said. “No explanation of how the proteins involved would work together to create crossover interference.”
Smith’s team unexpectedly came up with its solution to crossover interference while studying how the formation of DNA double-strand breaks (DSBs), the first step in crossover formation, are regulated.
DNA is made up of two complementary strands of molecules, and a split in both strands is referred to as a DSB. Crossovers are formed when the broken end of a maternal chromosome links up with the broken end of its paternal counterpart. (This also allows maternal and paternal chromosomes to increase genetic diversity by swapping large segments.)
Like crossovers, the frequency of DSBs is regulated during sex-cell formation. And both DSBs and crossovers occur at regularly spaced intervals along chromosomes, never getting too close together.
To examine DSB formation, Smith and co–first authors Kyle Fowler, then a research scientist in the Smith Lab (now a graduate student at the University of California, San Francisco), and Smith Lab research scientist Randy Hyppa turned to the yeast species Schizosaccharomyces pombe. The team looked at sites in the yeast’s DNA that have higher than average numbers of DSBs, known as DSB hotspots.
Hyppa and fellow author Dr. Gareth Cromie of the Pacific Northwest Research Institute found DSBs appear to “compete” with each other. If the researchers inserted into a chromosome a new DSB hotspot close to other hotspots, the frequency of DSBs at those other hotspots dropped. Conversely, removing a hotspot made the number of DSBs at nearby DSB hotspots increase. This DSB competition only reached a certain length along the chromosome.
The team also looked at a related phenomenon called DSB interference. The team saw that one DSB interferes with, or prevents, the formation of another DSB on the same chromosome, so that two DSBs rarely occur close to each other on one DNA molecule. Remarkably, this DSB interference spanned the same chromosome length as DSB competition.
An important clue to how competition and interference work came from looking closely at proteins called linear elements, which attach to chromosomes at hotspots. These proteins form distinct clumps when viewed under the microscope, and they must be present for DSBs to form at hotspots. Fowler found that the length of DNA in each cluster of linear elements roughly matched the distance over which DSB competition and interference act.
“We think that these foci [clumps under the microscope] correspond to physical clusters of hotspots,” Smith said.
Linear element proteins clustering hotspots together would explain how chromosomes “communicate” the presence of DSBs over long distances. Though hotspots are far apart along the 2D DNA molecule, gathering them together makes them near neighbors in 3D space.
This means that what happens at one hotspot can easily influence the others that are now nearby. The researchers found that another protein, called Tel1, appears to take advantage of hotspot closeness to repress both crossovers and DSB formation at nearby hotspots. Without Tel1, two DSBs formed at nearby hotspots much more often than expected. The team saw a similar change in crossovers without Tel1.
It appears that each cluster contains four to six hotspots (roughly the length over which DSB interference works). Smith theorizes that Tel1 senses the first DSB to form within a given cluster and likely (though this has yet to be shown) stems the tide of further breaks by turning off the activity of the proteins that create them.
Based on these findings, Smith proposes that the phenomenon of crossover interference arises from DSB interference.
“It just makes sense,” he said: Crossovers can’t form without a DSB, so molecular strategies that control how often (and where) DSBs form will likely also control how often (and where) crossovers form.
Smith’s team is currently delving deeper into the proteins involved in making the clusters and the nuances of crossover interference, including investigating what happens when the linear elements that create the hotspot clusters are removed. If these proteins are needed for crossover interference, then without them, the formation of one crossover should no longer interfere with the formation of others.
Though predicted, it’s not yet been shown that the clusters the researchers see occur only along the maternal or paternal chromosome, or whether hotspots from both the maternal and paternal chromosomes can group into the same cluster.
To help solve that mystery, Smith and his colleagues have turned to a particular yeast relative originally found in a bottle of fermented kombucha tea. When they cross this strain with their laboratory strain of S. pombe, slight variations in the kombucha-derived yeast’s DNA will help them trace the origin of each chromosome and their resulting clusters.
Time will tell whether the basics of the clustering mechanism, and Tel1’s integral role, hold true in other species. Smith acknowledges that the work is a long way from solving the problem of miscarriage or other medical issues that stem from improperly sorted chromosomes.
“But it does give a mechanism for how crossover interference is working. You can look through the history of medicine and see that knowing the mechanism of something is the first good step leading to either prevention or cure,” he said.
The National Institutes of Health funded this work.
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a Ph.D. in immunology from the University of Washington, an M.A. in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at firstname.lastname@example.org.