Downsizing DNA

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Marie Delattre, a biologist at the École Normale Supérieure de Lyon, has studied the nematode Mesorhabditis belari for nearly a decade now. The microscopic worm first caught her attention for its unconventional approach to reproduction, where only a small fraction of offspring keep their male parent’s DNA.¹ But more recently, she was looking at Mesorhabditis embryos under a microscope and noticed something else that was odd: in some embryos, the DNA was shattered into small fragments.

As she looked more closely across all stages of embryo development, she noticed a pattern. At the one-cell stage, the DNA looked normal. It stayed intact as the first cell divided into two cells and then three cells. But when the embryo reached the five-cell stage, the DNA fragments suddenly appeared.

They started in the nucleus, then moved into the surrounding cytoplasm, and after a few more rounds of cell division, disappeared entirely. Delattre was intrigued. During this brief point in the worms’ development, part of their genomes appeared to vanish.

In a wild-type cell, you would never see this. This would be a red flag.

 —Marie Delattre, Ecole Normale Superieure de Lyon

What she observed is a process carried out by dozens of other species. Termed programmed DNA elimination, this process allows organisms to delete specific portions of their genomes during development.  “It was a really random discovery,” said Delattre. “Serendipity, I would say.” Her team’s deeper investigation into this phenomenon, recently published in the journal Current Biology, added Mesorhabditis nematodes to the list of species that can carry out this mysterious process.^2,3

Even after more than a century of research, there are still many unanswered questions about programmed DNA elimination. 

“You see that this thing happens very commonly, suggesting it has an important biological role,” said Jianbin Wang, a biologist at the University of Tennessee, Knoxville. “It’s just that we don’t really know what that role is yet.”

Researchers around the world are working to answer these questions. Many of them stumbled upon programmed DNA elimination by accident, like Delattre, but now they’re hooked; their research could offer a new understanding of the ever-changing nature of the genome.

New technology takes on an old theory

An organism’s DNA starts off in one lonely cell containing the germline DNA passed down from the previous generation. Through many rounds of cell division, new somatic cells emerge that contain essentially identical copies of DNA and become the building blocks of the organism. 

In the 1880s at the Zoological Institute, cell biologist Theodor Boveri studied a species of parasitic worms called Parascaris, which has a relatively large genome compared to other worms—so large that its DNA was visible even through a primitive 19^th century microscope. He observed that a large chunk of the germline genome was removed as somatic cells developed.³ More than 100 years later, more sophisticated molecular biology assays revealed that this worm removed an astounding 89 percent of its 2.5-billion-base genome.

         

As those were still the early days of cell biology, Boveri assumed that this was a normal part of development. But as scientists looked for this process in more organisms, they realized that programmed DNA elimination was not universal. 

Early work focused on microscopic species, including various species of parasitic worms and single-celled organisms called ciliates. Scientists learned much of what they know about programmed DNA elimination by studying a family of parasitic worms called Ascaris, which removes around one-fifth of its germline genome.³ In the 1980s, researchers finally found a family of vertebrates, the hagfish, that removes between one-fifth and one-half of its germline genome.⁴ More recently, studies have shown that nearly all songbirds appear to eliminate parts of their germline genomes.⁵

“We are exposed to organisms that have programmed DNA elimination every single day,” said Alexander Suh, an evolutionary biologist at the Leibniz Institute for the Analysis of Biodiversity Change and Uppsala University.

Recent technologies such as DNA sequencing have bolstered researchers’ efforts to probe this process. By comparing sequences of the genomes of germ cells and somatic cells from the same organism, researchers can look for long stretches in the germline genome that are absent from the somatic genome. These studies have shown that species can eliminate anywhere between 0.5 percent and 90 percent of their genomes.

Scientists have used sequencing to investigate exactly which parts of the genome are removed and what instructions they encode. Typically, the same regions are removed in every cell of an organism and in every member of a species, although there are differences between species. However, regardless of species, the eliminated regions include large stretches of repeated DNA sequences, which typically do not encode the instructions for proteins.  

Many species undergo programmed DNA elimination, a process where specific parts of the genome found in the original sperm and egg cells are removed from the cells of the developing body. Different species use varied cellular mechanisms to remove specific parts of their genomes. This process has recently been documented in worms in the Mesorhabditis genus, which eliminate approximately thirty percent of their DNA.
Early in the process of Mesorhabditis development, cells still carry the germline genomes from the gametes that produced the first cell. As early as the two or four-cell stage, the DNA begins to fragment. Researchers can see this under a microscope, and it’s one of the first signs that a cell might be preparing for programmed DNA elimination.
As the cells prepare to divide, the DNA assembles into chromosomes. Normally, microtubules latch onto these chromosomes via each chromosome’s kinetochore proteins. However, some DNA fragments lack kinetochores, so the microtubules have nowhere to bind.
The chromosomes arrange themselves in pairs along the middle of the dividing cell in a region called the metaphase plate. Without microtubules to guide them there, the unattached DNA fragments do not migrate to the metaphase plate and instead linger in the surrounding areas of the cell. They will likely be targeted for elimination.
In the last stage of cell division, the microtubules pull the pairs of chromosomes apart, so that each new cell’s nucleus gets one chromosome from each pair. The unattached DNA fragments remain in the center of the dividing cell, where they will be randomly pushed into one of the two new cells.
Each new cell’s nucleus contains one full copy of the somatic genome, while the other DNA fragments remain in the cytoplasm outside the nucleus. Other species such as sea lampreys eliminate whole chromosomes instead of DNA fragments. During cell division, these chromosomes migrate to the metaphase plate along with the other chromosomes, but do not migrate to the poles of the dividing cell. This behavior is known as “lagging” and causes the chromosomes to be excluded from the new cells’ nuclei and eliminated.
After a few more cycles of cell division, the DNA excluded from the nucleus likely degrades in the cytoplasm, as seen in other worms.

How to ditch DNA

Programmed DNA elimination pops up on almost every branch of the tree of life, but the processes are as diverse as the flora and fauna that use them. The nematodes and unicellular ciliates seem to slice their genomes into small pieces and remove a subset. Vertebrates seem to be more likely to remove full chromosomes.

These different approaches involve the same set of core steps: part of the genome is marked for elimination, and as the cell divides, this DNA is shunted out of the nucleus and ultimately removed from the cell. In ciliates and worms, the cell also needs to slice up the DNA into fragments. How this happens in cells is still largely unknown. But studies from Wang, Delattre, and others are starting to piece together the process.

Cell division is a carefully orchestrated dance, where duplicated chromosomes pair up in a double-file line in the metaphase plate at the center of the cell. Long microtubules anchored at opposing ends of the cell latch onto the kinetochore protein at the center of each chromosome. As the cell divides, the microtubules reel in half of the chromosomes to each new cell. In Ascaris, Wang showed that DNA fragments that are ultimately eliminated actually lack kinetochore proteins, so the microtubules can’t bind to them and pull them into the new cells’ nuclei.⁶

Delattre saw a similar dearth of kinetochore proteins on eliminated DNA in Mesorhabditis.² Using a fluorescent label, she visualized DNA fragments as tiny dots floating in a ring around the rest of the DNA. The untethered DNA pieces get pushed to the perimeter while the remaining chromosomes segregate. As the cell continues dividing, the fluorescent dots end up outside the nucleus in the cytoplasm, and ultimately fade away. 

In vertebrate species, where whole chromosomes are eliminated, things go awry when microtubules start pulling the chromosomes apart. Certain chromosomes move slower than the others, a phenomenon called lagging. In a study of sea lampreys, a piece of DNA at the tip of the chromosome appeared to drag the chromosome in the opposite direction of the microtubule. As a result, these lagging chromosomes get left behind when the nuclei form and end up degraded in small pockets of the cytoplasm.⁷ 

Researchers still don’t know how the lagging chromosomes or discarded DNA fragments are chosen. In worms, Delattre uses detailed maps of the genome and RNA measurements to figure out how the cell knows where to fragment the DNA and what proteins make the cuts. Once she finds a compelling protein candidate, she hopes to delete it from an embryo and observe whether the cells still undergo programmed DNA elimination. 

Extreme DNA silencing

There’s another major open question: why would species want to eliminate their DNA at all? Removing DNA is an extreme way of keeping genes from being used in cells, according to Jeramiah Smith, a biologist at the University of Kentucky. But cells have other ways of doing this that are less disruptive. For example, they can pack their DNA so tightly that the genes can’t be accessed, or they can use small pieces of RNA that bind to genes and inhibit protein expression. Species that carry out programmed DNA elimination typically have the machinery to silence genes in these ways too.

Another explanation is that eliminated genes might play a role in germ cells during reproduction, where they may need to make an arsenal of proteins that are unnecessary in other cells of the body. Studies in Ascaris and in zebra finches revealed that their eliminated genes have functions in sex organs like the testes, where germ cells originate.^8,9

Understanding the origins of DNA elimination is going to tell us a lot about how our own genome works.

 —Jeramiah Smith, University of Kentucky

Not all researchers are convinced. In Mesorhabditis, Delattre showed that the eliminated genes seem to follow no pattern and aren’t critical to species survival.² She thinks that in these worms, the process could mainly serve to remove repeated sequences, and any genes that are removed are just bystanders.

It’s also possible that programmed DNA elimination could play diverse r oles in different species. “I would say, at this point, that they’re different processes,” Suh said. Smith agreed and noted that the evolutionary history of DNA elimination is still hazy. He speculated that each major branch of life may have independently developed the ability to eliminate DNA. “They’re doing similar things, but they got there through very different evolutionary trajectories,” he said.

Although programmed DNA elimination hasn’t been observed in humans, similar processes can occur in human cells, but typically only when they are malfunctioning. “In a wild-type cell, you would never see this,” Delattre said. “This would be a red flag.”

When human DNA breaks into fragments, a process called chromothripsis, it’s a cellular event so catastrophic that it can cause cancer. When human chromosomes don’t divide into cells properly during embryonic development, we end up with developmental disorders. “Understanding the origins of DNA elimination is going to tell us a lot about how our own genome works,” Smith said.

For example, it might help us better understand how these events occur in cancer cells. Knowing how DNA is marked for deletion and being able to do that artificially could even be used as a therapeutic option for disorders caused by extra copies of chromosomes, such as Down syndrome. 

For now, many scientists agree that the next step is to study programmed DNA elimination in more species. “We probably don’t know the majority of species that actually do this,” Smith said. 

Suh and his team are doing just that. So far, they have sequenced germline and somatic DNA from around 30 species of songbirds. Every songbird eliminates at least one chromosome, but in some cases it’s the biggest chromosome; in other species it’s the smallest one. The genes on the eliminated chromosomes can be completely different between species but they have one thing in common: Some of the gene sequences are very similar to those found on retained chromosomes, a trend that hasn’t yet been observed in other types of animals.⁹   

“It gets more and more confusing with every species,” Suh said.