ExpatSingapore - Darwin`s Mystery of myseries unravelled.

From New Scientist (long)

Darwin described the creation of new species as the "mystery of mysteries". Could the solution be found in a single gene?

ALTHOUGH Charles Darwin titled his book On the Origin of Species, speciation was one thing he could not explain. He called it the "mystery of mysteries", and even a century-and-a-half later the mechanism by which two groups of animals become genetically incompatible remains one of the greatest puzzles in biology. We understand how Darwin's Galapagos finches could have evolved from a single species - different populations became isolated and gradually adapted to different environments until they were no longer able to reproduce with each other. However, speciation also occurs rapidly and without physical isolation of populations, which is far harder to explain. Yet, amazingly, a single gene could hold the key.

That gene is at the heart of a crucial but seemingly irrelevant process called genetic recombination. During the production of eggs and sperm, chromosomes pair up, cross over and swap segments of DNA, mixing the genes you inherited from your mother and father (see diagram). This shuffling of the genetic pack is the source our individual uniqueness. But no one suspected that recombination might also play a role in generating new species.

The breakthrough came with the discovery that the gene that controls genetic exchange, which goes by the prosaic name of PR-domain containing 9, or Prdm9, is also implicated in generating reproductive incompatibility between different members of the same species. Dubbed "the speciation gene", if Prdm9's two roles turn out to be linked, it could be evolution's missing X factor.

The story of Prdm9 begins in 1974 with a young geneticist named Jiri Forejt at what was then the Czechoslovak Academy of Sciences in Prague. He was cross-breeding two subspecies of mouse, when he discovered that male offspring of certain combinations of parents were unable to reproduce. Further crosses indicated that differences in an unidentified gene were at least partly responsible for this reproductive isolation. In effect, the gene was creating two species. It was the first speciation gene known in vertebrates and still the only one known to cause "hybrid sterility". At that stage, though, its identity remained elusive.

Fast forward to the turn of the millennium, when Forejt and his colleagues finally tracked their speciation gene to a region on mouse chromosome 17 containing just six genes. Then, systematically ruling out the other five, they cornered their culprit - a gene called Prdm9 (Science, vol 323, p 373).

Meanwhile, at the University of Oxford, evolutionary biologist Chris Ponting had begun what seemed like a totally unrelated research project: rummaging through the human genome in search of the genes that make us unique. "If you want to know what makes humans human, you need to find out which gene has evolved fastest in humans," Ponting says. To his surprise, he came up with the same gene Forejt had tagged just a few months earlier - Prdm9 (PLoS Genetics, DOI: 10.1371/journal.pgen.1000753). "It has the claim of being the most rapidly evolving gene in human history," says Ponting. Humans and chimps differ at more than 7 per cent of the DNA letters in Prdm9, which is over five times the average difference between the species.

Why, Ponting wondered, should his search for an evolutionary sprinter turn up a so-called speciation gene, rather than one affecting some uniquely human trait such as language or a large brain? When he looked more widely, the answer became apparent: Prdm9 evolves with extraordinary rapidity throughout the animal kingdom, from rodents and sea anemones to snails and worms. It is not special to humans at all.

Ponting's search for human uniqueness had hit a dead end. But Forejt's esoteric mouse gene was starting to look more and more interesting. Here was a gene with a clear evolutionary role, yet also linked to sterility, an evolutionary dead end. The key to resolving the contradiction surely lay in finding out Prdm9's function. "No gene evolves to make sterile mice," says Michael Nachman, an evolutionary geneticist at the University of Arizona. "Prdm9's role in hybrid sterility is an accidental by-product of its proper role." But what was that role? The answer soon came, from yet another direction.

Genetic recombination was first described a century ago, but research had not moved on much since 1931 when geneticist and Nobel laureate Barbara McClintock demonstrated the mechanism of crossing over. However, the ability to sequence entire genomes sparked renewed interest in recombination, particularly in the question of where exactly crossover occurs. It had been assumed that it happens randomly across the genome, but closer inspection revealed that at least 80 per cent of crossover events happen at so-called "hotspots" of recombination. (There are at least 25,000 in the human genome, although when an egg or sperm is produced only a tiny fraction of these is active.) This arrangement makes sense, if hotspots exist to channel crossovers away from crucial parts of the genome. "Recombination involves breaking DNA, and when you mend DNA you sometimes make mistakes," says Gil McVean, a statistical geneticist at the University of Oxford. So sticking hotspots near but not in genes could certainly be seen as a good thing."

Then, in 2008, McVean and his colleague Simon Myers discovered that about 40 per cent of hotspots have the same 13-letter DNA sequence, or motif, and they realised that something probably homes in on that motif to activate the hotspot and trigger recombination. The most likely candidate for the job would be a so-called zinc-finger protein - a key-like molecule that recognises and binds to particular DNA sequences, usually to initiate the process of transcription.

Last year, three research groups independently identified the protein involved. It was indeed a zinc-finger protein, according to teams led by McVean, Bernard de Massy at the Institute of Human Genetics in Montpellier, France, and Kenneth Paigen at the Jackson Laboratory in Bar Harbor, Maine. And the gene responsible for making it? You guessed it - our friend Prdm9 (Science, vol 327, pp 835, 836 and 876, respectively). Taken together, the three studies made a strong case that Prdm9 plays a crucial role in activating recombination hotspots, at least in the 40 per cent that contain McVean's 13-letter motif. Most researchers who have anything to do with Prdm9 think it probably plays a more ubiquitous role and may recognise other hotspot motifs as well, although no others have been identified as yet.

The discovery of Prdm9's function can at least shed light on why it evolves so rapidly. Each time a crossover occurs, part of the DNA sequence around the break is lost, erasing the hotspot motif on the chromosome to which the Prdm9 protein is bound. The cell patches this hole by copying the sequence from the undamaged region of the other chromosome of the pair. But occasionally an individual carries the hotspot motif at that location on only one chromosome, and the repair erases the hotspot. So as eggs and sperm are produced, and time goes on, hotspots should gradually wink out, one by one, until none remain. Obviously, that does not happen, and Prdm9 provides the reason: while mutations in most genes tend to be damaging, any mutation that changes the Prdm9 protein's zinc fingers will simply alter the sequence of DNA to which it binds, gradually creating new hotspot motifs. "You need recombination, so you don't want to be eroding motifs," says McVean. "But if you've got a gene that is constantly changing what it binds to, erosion won't be a problem. You'll be moving motifs before it has caught up with you."

Moreover, Prdm9 is built for change. The protein it codes for has several zinc fingers - 12 or 13 in most human versions - whose coding sequences line up one after the other in a structure called a minisatellite. For reasons that are not yet completely understood, minisatellites are unusually prone to mutation, so Prdm9 seems well equipped to hold up its end of the evolutionary race against eroding hotspots. In fact, McVean speculates - but cannot yet prove - that the zinc fingers of the Prdm9 protein may bind to motifs within the Prdm9 gene itself, making it a recombination hotspot in its own right, and so even more prone to rapid evolution.

All this paints a very busy picture of the genome. In a time-lapse sequence running over hundreds or thousands of generations, hotspots would constantly be blinking on and off throughout the genome, with Prdm9 as the engine driving this incessant drumbeat of change. At the very least, this means that individuals who carry different variants of the Prdm9 gene will differ in the hotspots they use, and perhaps even in the recombination strategies they employ (see "Ways to shuffle a genome"). McVean notes that this could also affect the sorts of genetic diseases to which different people are prone, since errors in recombination resulting in accidental duplication or deletion of DNA often cause specific diseases such as Charcot-Marie-Tooth disease and certain hereditary neuropathies.

What's the link?
So Prdm9's role in recombination can explain its very rapid evolution, but what about the link with speciation? Does specifying the points at which chromosomes swap material somehow give Prdm9 the power to make certain combinations of egg and sperm incompatible, or are these two, independent abilities combined in one gene by sheer coincidence? Most Prdm9 researchers lean towards the first explanation. "It makes sense that there must be some common mechanism," says Forejt.

Mice with incompatible variants of Prdm9 are clearly sterile, but there is some way to go before we know whether this has anything to do with recombination. As yet, nobody has even looked for a link between Prdm9 and sterility in species other than mice. We do know, however, that different lineages of humans sometimes carry different variants of Prdm9. In most Europeans, for example, the Prdm9 protein has 13 zinc fingers, but the number ranges from eight to 18. In Forejt's mice, differences like that can be enough to produce sterile offspring. Might the same be true of humans? Ponting thinks it is worth investigating. "Variation in this gene could be driving a wedge between different parts of our human population," he says.

It is an intriguing idea, but the evidence to date seems not to corroborate it. Alec Jeffreys, the inventor of DNA fingerprinting, and colleagues at the University of Leicester, UK, compared the sperm count of men who had inherited the same version of Prdm9 from each parent with that of men who had inherited different versions. The "hybrid" men showed no tendency to lower sperm counts, as estimated by DNA yield from sperm samples, says team member Ingrid Berg.

Of course sperm count is not the only measure of fertility, and may be irrelevant in understanding the link between Prdm9 and hybrid sterility. The trouble is that it's not obvious why two individuals who differ in their recombination hotspots might be sexually incompatible. Continuing research with mice may help to solve the problem by revealing the interactions between Prdm9 and other genes. Although Prdm9 seems to be key, no gene works entirely in isolation and the new research should give a better picture of what is going on at the genetic level. Forejt is close to pinpointing one such gene, located on the X chromosome, and there is another in the frame, although its identity remains unknown. Once these other actors are understood, it may be easier to understand whether, and how, Prdm9 contributes to hybrid sterility in other species, including humans.

"We can speculate that this could be some sort of universal reproductive-isolation gene in animals, which would be beautiful," says Forejt. If that turns out to be the case, Prdm9 would be a key to understanding how one species can split into two - Darwin's mystery of mysteries. Then the gene responsible for the seemingly innocuous process of genetic crossover truly would deserve to win the title of evolution's X factor.

Ways to shuffle a genome
Whenever an animal produces an egg or sperm, it first shuffles the genetic pack in a process known as recombination (see diagram). This has the advantage of creating genetically diverse offspring, increasing the likelihood that at least some of them will survive. It is also what makes each of us - except identical twins - unique.

One surprising discovery is that some people do a more thorough job of shuffling than others. "There are people who recombine a lot before they hand their genomes to their offspring, and there are those who recombine little," says Kari Stefansson, CEO and founder of deCode Genetics in Reykjavik, Iceland. What's more, such differences extend to entire populations. For example, low levels of recombination are more common in people of African descent - who are more genetically diverse to begin with - than in Europeans. This suggests that evolution strikes a balance between the benefits of genetic diversity and the risk of introducing genetic errors through recombination.

Last year, Stefansson and his colleagues reported intriguing evidence that men and women, too, may have different recombination strategies (Nature, vol 467, p 1099). They used detailed genomic data from more than 15,000 parent-offspring pairs to track exactly where on the genome recombination events had occurred between the generations. They found that in women the crossover points tended to occur between genes, thus producing new combinations of genes. In men, the hotspots were slightly more likely to occur within spacer regions, or introns, which separate parts of a single gene, allowing recombination to create new versions of genes. No one knows why the two sexes should employ these different strategies but, once again, it looks as though males take the more risky approach even when it comes to shuffling their genetic pack.