Mon. Nov 28th, 2022
Image of pieces of DNA labeled with fluorescent tags.
enlarge / Individual chromosomes (right) have a complex organization when packed into the nucleus of a cell (left).

Complex organisms have complex genomes. While bacteria and archaea keep all their genes on a single DNA loop, humans spread them across 23 large DNA molecules called chromosomes; The number of chromosomes varies from a single chromosome in males of an ant species to more than 400 in a butterfly.

There is some evidence that chromosomes are important for the underlying biology of an organism. Specialized structures in it affect the activity of nearby genes. And studies show that regions on different chromosomes will consistently be found next to each other in the cell, suggesting that their interactions are significant.

So how can we square these two facts? The number of chromosomes varies greatly and sometimes differs between closely related species, suggesting that the actual number of chromosomes does not matter much. Still, the chromosomes themselves appear to be critical for an organism’s genome to function as expected. To investigate this problem, two different groups tried a daring experiment: Using genome editing, they gradually merged the 16 chromosomes of a yeast into just one giant molecule. And unexpectedly, the yeast was mostly fine.

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Combining the yeast’s 16 chromosomes involved repeated applications of the same process. Chromosomes are simply large molecules of DNA, and they have two structures that need to be managed. The first is at each of their two ends, where there is a structure called a telomere. The loose ends of the chromosome are difficult to copy when a cell divides, are susceptible to damage, and can trigger the cell’s response to dangerous damage to DNA. The telomere handles all of that, protecting the ends and providing a mechanism for their duplication.

The other problem is something called a centromere. This can be located anywhere in the chromosome and plays a crucial role when cells divide. It keeps both copies of the chromosome linked together and is then where fibers are attached that pull the two chromosomes apart, one in each daughter cell. Having two centromeres on a single chromosome is a problem because fibers can attach in such a way that a single chromosome is pulled towards both daughter cells, causing it to fall apart.

So to combine two chromosomes, you have to chop off two of the telomeres, fuse the new ends together to form a single chromosome, and chop out one of the centromeres.

Cutting things up is exactly what CRISPR DNA editing technology is good at. When designing their editing system, the researchers directed the CRISPR proteins to specific locations near telomeres, where they cut the chromosomes. Extra DNA molecules they put in these cells promoted a repair of the cuts that fused the cut ends of the chromosomes together. Additional CRISPR targeting would exclude one of the centromeres. The fusion of chromosome pairs reduces the 16 chromosomes to eight. Further mergers could continue to reduce the overall number.

In one case, the researchers continued this process down to a single giant chromosome. But the second group got stuck at two; attempts to combine them continued to kill the cells. The researchers think they know why this happened, and we’ll get to that in a moment.

Remarkably healthy

The resulting yeast strains (with only one or two chromosomes, depending on the lab) were… remarkably normal. They grew more slowly than a normal species, but still grew under different conditions. They were more sensitive to how they obtained critical building blocks, such as nitrogen and carbon, but could still survive no matter how they were fed. And tests of their gene activity showed very minimal changes compared to a wild-type strain – just 28 genes out of more than 5,800. The normally structured arrangement of DNA in the cell was completely disrupted, but this did not appear to affect the health of the yeast.

The big problems came with mating (yes, yeast has sex). The default state of yeast is to carry a single set of chromosomes. When they mate, two yeasts fuse together, creating a single yeast with two sets of chromosomes. This can divide and keep itself in this state with two chromosomes, or it can form spores, each of which has a single set of chromosomes. Tests showed that although cells with a single chromosome genome could mate and form spores, the viability of these spores was lower than in a normal strain.

Attempts to mate normal strains with strains that carry fewer chromosomes resulted in very low fertility, as the chromosomes would have a hard time lining up to be distributed normally to daughter cells. The researchers suggested this could be helpful; performing genetic engineering on cells with fewer chromosomes would mean that the engineered genes would not mix with the yeast population if it escaped into the wild (or a bakery).

So, why do these yeasts have problems? One clue is that the single huge chromosome makes DNA copying and DNA separation difficult during cell division. Although very few genes saw altered activity, a number of the genes that were altered are involved in stress responses.

A second problem comes from changing the number of telomeres at the ends of chromosomes. Normally, genes near telomeres have decreased or stopped their activity; yeast tends to have nearby genes that are only needed under specific environmental conditions. Some of these genes have ended up in the center of the giant chromosome through all the DNA editing and may not be properly regulated as a result. In addition, the fact that there were only two telomeres means that the proteins that normally shut down nearby genes were in excess, allowing the gene shutdown effect to spread further from the two remaining telomeres. In other words, some genes that would normally be far enough away from the telomeres may have been turned off on the giant chromosome.

All of this may explain the yeast’s slower growth rate and problems with certain specific environmental conditions. It may also explain why some specific combinations of chromosome fusions failed, leaving one research group in two stuck. The beauty, however, is that these problems must be subject to evolutionary change. By growing the single chromosome-carrying yeast in culture for many generations, we should see them adapt to their foreign genome. And those changes may give us some indications of which aspects of chromosome biology are critical to normal genome activity, and which are just historical coincidences.

Nature2017. DOI: 10.1038/s41586-018-0382-x, 10.1038/s41586-018-0374-x (About DOIs).

By akfire1

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