Frozen mammoth skin retained its chromosome structure

One of the challenges of working with ancient DNA samples is that damage accumulates over time, breaking up the structure of the double helix into ever smaller fragments. In the samples we've worked with, these fragments scatter and mix with contaminants, making reconstructing a genome a large technical challenge.

But a dramatic paper released on Thursday shows that this isn't always true. Damage does create progressively smaller fragments of DNA over time. But, if they're trapped in the right sort of material, they'll stay right where they are, essentially preserving some key features of ancient chromosomes. Researchers have now used that to detail the chromosome structure of mammoths, with some implications for how these mammals regulated some key genes.

DNA meets Hi-C

The backbone of DNA's double helix consists of alternating sugars and phosphates, chemically linked together (the bases of DNA are chemically linked to these sugars). Damage from things like radiation can break these chemical linkages, with fragmentation increasing over time. When samples reach the age of something like a Neanderthal, very few fragments are longer than 100 base pairs. Since chromosomes are millions of base pairs long, it was thought that this would inevitably destroy their structure, as many of the fragments would simply diffuse away.

But that will only be true if the medium they're in allows diffusion. And some scientists suspected that permafrost, which preserves the tissue of some now-extinct Arctic animals, might block that diffusion. So, they set out to test this using mammoth tissues, obtained from a sample termed YakInf that's roughly 50,000 years old.

The challenge is that the molecular techniques we use to probe chromosomes take place in liquid solutions, where fragments would just drift away from each other in any case. So, the team focused on an approach termed Hi-C, which specifically preserves information about which bits of DNA were close to each other. It does this by exposing chromosomes to a chemical that will link any pieces of DNA that are close physical proximity. So, even if those pieces are fragments, they'll be stuck to each other by the time they end up in a liquid solution.

A few enzymes are then used to convert these linked molecules to a single piece of DNA, which is then sequenced. This data, which will contain sequence information from two different parts of the genome, then tells us that those parts were once close to each other inside a cell.

Interpreting Hi-C

On its own, a single bit of data like this isn't especially interesting; two bits of genome might end up next to each other at random. But when you have millions of bits of data like this, you can start to construct a map of how the genome is structured.

There are two basic rules governing the pattern of interactions we'd expect to see. The first is that interactions within a chromosome are going to be more common than interactions between two chromosomes. And, within a chromosome, parts that are physically closer to each other on the molecule are more likely to interact than those that are farther apart.

So, if you are looking at a specific segment of, say, chromosome 12, most of the locations Hi-C will find it interacting with will also be on chromosome 12. And the frequency of interactions will go up as you move to sequences that are ever closer to the one you're interested in.

On its own, you can use Hi-C to help reconstruct a chromosome even if you start with nothing but fragments. But the exceptions to the expected pattern also tell us things about biology. For example, genes that are active tend to be on loops of DNA, with the two ends of the loop held together by proteins; the same is true for inactive genes. Interactions within these loops tend to be more frequent than interactions between them, subtly altering the frequency with which two fragments end up linked together during Hi-C.

Mammoth challenges

That's how Hi-C works in theory. Putting it into practice on a mammoth posed a number of pretty significant challenges. To start with, it's far easier to interpret Hi-C data if you already know what the genome should look like. Unfortunately, we only had a limited idea in the case of mammoths. The mammoth genome itself was highly fragmented due to its origin in ancient DNA. But even the genomes of its living relatives, the African and Asian elephants, are highly fragmented, since they were done a few years back using a quicker and inexpensive approach. So, as a first step, the researchers generated a high-quality genome for both the living species, including generating Hi-C data from them.

Hi-C data was generated from the mammoth by repeating the procedure 26 times, producing 4.4 billion sequence fragments from linked pieces of DNA. As is often the case with ancient DNA, most of these were contaminants—in this case, 97 percent were. After getting rid of data from neighboring fragments that had been linked back together, a total of 4.6 million fragments were useful for Hi-C analysis. That's only 0.1 percent of the original data; when Hi-C was done with Asian elephant cells, the corresponding figure was 43 percent, illustrating the scale of the challenge when working with ancient DNA.

That said, this sequence appeared to be the real deal. Changes in the base sequence that occur due to damage were elevated, and individual base differences were almost always the same as those seen in mammoths. There wasn't enough mammoth data to do the full Hi-C analysis from scratch, so the researchers focused on identifying differences between mammoth and elephant data. If there wasn't an indication of differences, the mammoth's chromosomes were assumed to look like an elephant's.

Critically, the results showed that the frequency of contact vs. distance along the chromosome relationship for the mammoth DNA was the same as that seen in Asian elephants. This indicates that the basic chromosome structure had been preserved over 50,000 years in ice, even as the underlying chromosome had decayed into ever-smaller fragments.

The resulting mammoth genome had the same total number of chromosomes as modern elephants, and large-scale rearrangements, like duplicated or flipped sequences, were infrequent. The data was also sufficient to pick out some of the loops that occur around active and inactive genes. The mammoth sample had come from skin tissue, and the pattern of looping was similar to that seen in elephant skin. In fact, data from elephant skin was more similar to mammoth skin than it was to any other elephant tissue tested.

There were a few differences, however, including a couple of genes that are known to be involved in hair production in other mammals. It's worth noting, however, that not all of these changes in gene activity were what you'd expect to be needed to produce more hair in the mammoth, suggesting that its shaggy coat was the result of a more complicated evolutionary path than "turn the hair genes on." That's consistent with the fact that there were over 800 differences in gene regulation identified in total.

Data from the X chromosome also had indications that one of the animal's two Xes was inactivated, indicating that it was a female.

Skin-deep

Based on the size of DNA fragments typically found in the mammoth genome, the researchers estimate that chromosome features down to roughly 50 nanometers had been preserved and that it was likely that many even smaller than that were still present in this sample. They suspect that the extremely cold, dry air of its habitat had essentially freeze-dried the chromosomes in place, something that might occur in other samples preserved in the Arctic or in dry environments with more moderate temperatures. (They tested the latter using beef.)

While the findings are impressive, it's important to highlight that this isn't a revolution in our understanding of the mammoth. Due to its close relationship with existing elephants, we'd have expected its genome to look and operate fairly similarly. And this has only told us about gene activity in skin cells; while those have some distinct adaptations in the mammoth, they're only one of a long list of tissues that would have needed to change to adapt to its very different diet and habitat.

That little dose of perspective, however, should not stop you from being amazed that we are able to obtain this sort of information from an animal that has been dead for 50,000 years.

Cell, 2024. DOI: 10.1016/j.cell.2024.06.002  (About DOIs).