The “Y” Files

Paradigm magazine | Fall 2003
By Kelli Whitlock

 

Not everyone believes in chance—the fortuitous twist of fate that takes a person down an unforeseen path. David Page believes in chance. For him, fate came along on the tip of a light brown wooden toothpick.

Page was a first-year medical stu-dent and research assistant in Cambridge, Massachusetts, in 1979 when he signed up for a study to map all the genes in the human body. (Seven years later, this effort would become known as the Human Genome Project.) His first task was to analyze thousands of recombinant DNA clones, searching for those most suitable for study. Each clone was selected randomly, and rather unceremoniously, from a sea of thousands. The instrument used for this scientific experiment: a toothpick.

It was summertime and Page was spending hours in the lab, waving a toothpick above trays of DNA clones, choosing one, examining it, and choosing another. One after-noon in particular stands out in his memory. What ended up on the bottom of his toothpick that day was a clone of DNA shared by the X and Y chromosomes—the sex chromosomes whose combination determines whether a human is male or female. He examined the clone, pondering its potential for his project, just as he had done with dozens of others. It’s only in hind-sight, 24 years later, that the magni-tude of that random selection became clear.

“When people ask how I picked the Y chromosome for my research, I often say, ‘I didn’t. It picked me,’” Page says. “If I hadn’t picked that clone out of hundreds of thousands I’d probably be a cardiologist.”

Not a cardiologist, but a scientist at Whitehead Institute and an investigator with Howard Hughes Medical Institute, Page is considered to be among the world’s leading experts on the Y chromosome, the defining biological determinant that makes males male. In 1992, his lab announced the first successful cloning of a human chromosome—the Y chromosome—and in June of this year, he led a team that published the complete sequence of the Y on the cover of Nature, work that offers not only a road map for scientists who study male infertility, but also casts doubt on a decades-old theory that destined the Y to extinction.

And it all began with a fateful stab of a tiny wooden stick.

 

The Y’s Demise

Scientists marvel at the nuances of the human genome, the mysterious alphabetical configurations of DNA and the construction of cells and genes and chromosomes that work together to help us live, reproduce, and evolve. At the heart of human evolution is the body’s ability to repair genetic flaws through a process called sexual recombination.

All humans receive a set of chromosomes from Mom and a matching set from Dad. Over the course of many generations, chromosomal pairs can swap damaged genes for good ones and fill in gene sequences that may be missing on one chromosome but present on its mate. This swap—called recombination—doesn’t fix all damaged or missing gene sections. It’s up to natural selection to eliminate those that make it through without repair.

If all chromosomes had a matching partner, the story would end here. But the complexities of human biology make things messy. Of the 24 chromosomes in the human genome, 22 come in identical pairs in both males and females. Women have another matching set—two X chromosomes that together cast a developing fetus in a female role. But men have a mismatched arrangement of sex chromosomes—one X and one Y. Lacking a mate, the Y can’t swap its defective genes for good ones.

The Y has paid dearly for this bachelor status over time. When sex chromosomes first evolved some 300 million years ago, the X and Y each had about 1,000 genes, which they swapped with each other.

Somewhere along the way, the Y lost its ability to share genes with the X. As defects in the genetic structure appeared, the Y was stuck with them and most of the chromosome’s genes weakened or died out altogether.

Indeed, in the biological battle between the sexes, the Y chromosome has suffered defeat after defeat. The male-determinant has seen its gene supply shrink to what scientists thought was only a handful of genes; some speculated that there was just one lone gene on the Y—the one responsible for maleness. It was a downward trend predicted to continue until the Y disappeared altogether. 

 

X marks the spot…or does it?

David Page is a lone Y in a house full of Xs. He and his wife Elizabeth have three daughters, an irony not lost on a man whose research subject often provides fodder for only half-feigned derogatory sarcasm from the double-X gender. The Y chromosome long has been the whipping post for all stereotypical male traits—all negative stereo-typical male traits—including everything from an inability to ask for directions to the bewildering memory skill that allows for instant recall of exact dates of historical sporting events but not dates of anniversaries and birthdays.

Subscribing to the “If you can’t beat ‘em, join ’’‘em” philosophy, Page’s research presentations often include a slide featuring certain “genes” identified along the Y chromosome: the genes for channel flipping (FLP), spitting (P2E), the ability to identify aircraft from a distance of 10,000 feet (DC10), and selective hearing (HUH?).

Obviously, the 47-year-old Pennsylvania native has a healthy sense of humor. He also has a fondness for treading on uncharted territory. As a high school senior in a small rural town near Three Mile Island, Page applied to Swarthmore College, a private, liberal-arts college, while many of his other classmates chose to remain closer to home. He was accepted and enrolled as a freshman intent on a career in environmental law. While Page enjoyed a rich exposure to chemistry, physics, and biology in high school (he calls the experience a “Sputnik education,” sparked by the nation’s 1950s desires to outpace the Russians in space and science), he also enjoyed the debate team, and law seemed a natural step.

“When I was growing up, science was very much an abstraction because I’d never met a scientist. I had no idea what a scientist looked like or, for that matter, if anyone actually was a scientist,” Page recalls. It took a while for the science to take hold, but by his junior year at Swarthmore, he “came back to the Sputnik stuff.”

Page spent two summers as a research assistant in biology labs, first at Brookhaven National Laboratory on Long Island, New York, and then at the National Institutes of Health in Bethesda, Maryland. At NIH, he studied nucleosomes, basic subunits of the chromosome. He was hooked.

He applied to the Health Sciences and Technology Program, a joint initiative between Harvard University and Massachusetts Institute of Technology that integrates education and research in science, engineering, and medicine. To fulfill a research thesis requirement, he joined the lab of David Botstein at MIT, a pioneer of the Human Genome Project who now leads Princeton University’s genomics institute. It was there that Page had the chance meeting with a gene clone shared by the X and Y. The Y wasn’t all that interesting to genetic scientists at the time. Seemed like a perfect fit for a man who enjoyed forging paths rather than following them, so when Page received his MD and joined Whitehead as a Fellow in 1984, he continued the work he began at MIT—a project to map the Y’s gene sequence.

 

Making a map

In 1992, four years after being named an Associate Member at the Institute, Page’s lab cloned the Y chromosome—the first time anyone had cloned a human chromosome. Over the next 10 years, the scientist’s work revealed new information about the evolution of the Y and the function of its genes. In the late 1990s, the biologist and his collaborators published findings that suggested that infertile men who father children through a common type of in vitro fertilization can pass along to their male offspring the very genetic flaws that caused their own infertility.

But the biggest advance—the completion of the Y mapping project—was announced at a Washington, D.C., press conference in June 2003. The effort, led by Page and collaborators from Washington University School of Medicine in St. Louis, yielded 78 genes on the chromosome—far more than the handful rumored to remain on what had come to be called the “rotting Y.”

And there’s more: While it’s true that over millions of years the male sex chromosome has lost hundreds of genes and seen many others crippled, the biggest concern has been gene health in the regions of the Y that control sperm production. But this new genetic map reveals a series of massive palindromes—stretches of gene copies that are 99.9 percent identical to one another. A palindrome is something that reads the same forward and backward (i.e., MADAM I’M ADAM), and the researchers found eight of them in the region of the Y responsible for sperm production. The scientists suspect that this genetic “hall of mirrors” provides a mechanism for self-repair, a way for the Y to prevent the erosion of these critically important genes.

Technology has not yet provided a window to watch the chromosome in action, which leaves the researchers to infer the function of these duplicate gene sequences. Say a gene copy along one of these palindromes suffers a mutation. By bending into a hairpin formation, the injured gene pairs with its copy, and the good gene may overwrite the bad one. Essentially, the Y combines with itself.

“This study shows that the Y chromosome has become very efficient at preserving its important genes,” says Richard K. Wilson, director of the Genome Sequencing Center at Washington University School of Medicine in St. Louis, where the Y was sequenced. “It’s found different ways to do the things chromosomes must do to evolve, survive, and thrive.”

But this secret weapon was not revealed easily. While other chromosomes are known to have duplicate genetic sequences, none contains quite as many. Wilson’s team recently completed sequencing chromosome 7, a task he considered among the biggest challenges his lab has tackled. Duplicate sequences constitute about 8 percent of that chromosome; they make up half of the Y.

“There are some things that just don’t like to be sequenced,” Wilson says. “They can be a bit resistant to being deciphered by the usual biochemistry methods we use. So, we had to use some alternative biochemistry for the Y.”

Researchers mapped the gene sequence of a Y chromosome from an anonymous male, as well as parts of a Y chromosome from a chimpanzee. This technically challenging process involved delicately unwrapping the two arms on each of the eight palindromes and analyzing the near-identical gene sequences inside.

“Most chromosomes are like a typical thousand-piece jigsaw puzzle—a pretty picture split into pieces with easily identifiable markings,” says Wilson. “The Y chromosome, on the other hand, was like a picture of a small sailboat on the ocean with lots of blue sky, no clouds, and hundreds of pieces that looked exactly alike. Determining exactly where each piece went in the grand scheme required a lot of work.”

Key to these findings is that researchers identified this gene repair technique not only in a human Y chromosome, but also in a chimp Y.

“When we look at the human Y, compared with the chimp Y,” Page says, “what we can infer is that during the last 5 million years, since we and chimps parted company, this overwriting of one gene copy by another has been going on frequently in our Y chromosome and in the chimp Y chromosome.”

 

Questions answered, questions raised

Studies of the Y chromosome in humans and other species haven’t always caught the collective eye of biologists. In fact, the Y chromosome has not been studied in com-parable detail in any other species.

But the small number of people interested in the Y has steadily increased in the last few years. Today, the field is populated with researchers interested in a variety of projects in which the Y chromosome is implicated, including the mystery surrounding the origins of modern populations (called the search for Y-Chromosomal Adam) and male infertility.

Millions of couples in the United States alone have trouble conceiving a child. In about 30 percent of those cases, the problem is related only to male infertility. Steve Rozen and Helen Skaletsky, scientists in the Page lab and coauthors of the Nature studies, are interested in applying the information they’ve learned about the Y’s genetic make-up to their male infertility studies. Of all the genes the team identified on the chromosome, all but 18 are active in the testes, Rozen says.

“Some of these genes are essential for normal sperm production. For others, the fact that the gene is active in the testes merely suggests a role in sperm production,” Rozen notes. “We are interested in looking for damage to these genes in men who do not produce normal numbers of sperm. Finding a newly damaged gene in a man with poor sperm production tells us that the damage caused the sperm production problems.”

Understanding the structure of the Y chromosome may be a significant step forward in the effort to treat male infertility. But, Rozen cautions, science moves at its own pace, which is hardly ever fast.

For some scientists, though, the pay-off of this work is more immediate. 

“I thought about, talked about, wrote about the Y as a rotting chromosome that really only had one important gene—the one that determines sex,” says Scott Hawley, a biologist with the Stowers Institute for Medical Research in Kansas City, Missouri, who studies chromosomal pairing. But these new findings make “perfectly good sense,” Hawley adds. “It’s one of those ‘Ah-ha!’ experiences that, after you hear it, you think, ‘It had to be that way. Why didn’t we think of this before?’ It’s just revolutionary work.”

The research could perhaps have the most significant effect on studies of heterochromatin, highly condensed chromatin (portion of a cell nucleus that contains all the nucleus’s DNA) strains once thought to be useless genetic wastelands but now known to be essential for normal chromosomal behavior.

“David’s really given us an analytical approach to studying heterochromatic regions on a larger scale,” says Hawley, who authored a review of the Page lab findings for the journal Cell. “It’s a new paradigm for thinking about the structure of the heterochromatic regions, and there are a lot of people who think about this kind of stuff.”

 

Getting a little respect

“I often say that the Y chromosome is the Rodney Dangerfield of the chromosome world,” Page jokes. “It gets no respect.”

No respect and little credence: When the completion of the mouse genome was announced in 2002, it was not really complete. The mouse Y has not yet been sequenced.

A 2002 article in Nature by two Australian scientists rang a death knell for the Y chromosome, claiming that “The original Y chromosome contained around 1,500 genes, but during the ensuing 300 million years, all but about 50 were inactivated or lost… . At the present rate of decay, the Y chromosome will self-destruct in around 10 million years.”

There’s no denying the Y has problems. While this new research shows that there are more genes on the chromosome than once thought, as Page points out, the Y still has lost a lot of genes. But to expend its energy on protecting the genes that are most important, the ones that keep it from extinction—now that, Page says, is clever.

“The Y has found a way to keep these genes coherent despite a rather unstable structure,” says Robert Waterston, now a scientist at the University of Washington who was a lead researcher at the Genome Sequencing Center at Washington University during these studies. “That instability of structure could be disastrous for a particular individual, but it won’t be disastrous for the Y, because the deleted Ys would not be passed on.”

Still, not everyone is convinced that this justifies a newfound respect for the Y chromosome. The oft-targeted male chromosome took a thrashing in a column published in early July in the Boston Globe by David Bainbridge, a fellow of St. Catharine’s College in Cambridge, United Kingdom, and author of The X in Sex: How the X Chromosome Controls our Lives.

“Page’s research appears to demonstrate that the Y chromosome contains clear evidence of trying to patch up its wounds by swapping bits with itself. Of course, this is still an inefficient way of losing damaged genes. It may hold things together in the short run, but it’s no healthy way for a self-respecting chromosome to carry on,” Bainbridge wrote.

Attacks such as this confound Page at times. He acknowledges that the Y likely will always be the butt of many jokes. Still, when the abuse is heaped on by other scientists, it gives him pause.

“The common perception of boys and girls, of men and women, greatly impacts biologists’ perceptions of the X and the Y,” Page says. “The science and the sexual politics become blurred. The idea of the Y as a shiftless, no-good degenerate chromosome is entirely too appealing and attractive to resist for reasons that have little do with science and lots to do with sexual politics.”

 

The road ahead

Over the last two decades, the Y chromosome has revealed itself to be far more complicated than any-one thought. What many scientists overlooked for other, seemingly sexier research topics has provided Page with a biological challenge that is far from over.

“There are many points in the course of a line of experiments where you choose to believe or dis-believe something and you choose to follow it up or let it go,” he says. “The history of Y chromosome research is strewn with prematurely abandoned lines of work.”

But there was something about the chromosome that demanded Page’s attention. As the sequence of the Y was coming together, scientists learned bits and pieces about the chromosome. But until the map was complete, Page says, they were fum-bling along in the dark. Now, the team will use the human Y as a reference for the study of the Y chromosomes of other organisms.

Already under way are projects to sequence the male-determining chromosome in the chimpanzee and the mouse. The chimp Y should be complete in 2004 and the mouse Y the following year.

“We’ve seen the Y in humans and we’ve started to see little bits in the chimp and we’re beginning to see that there’s something a little different,” Wilson says. Filling in those missing pieces would go a long way toward understanding how sex chromosomes have evolved and learning why different organisms choose the specific type of reproductive strategy they use.

Of course, the studies of the Y’s genetic self-repair system will continue, as will plans to identify how genes in the testes region function, and what happens when they don’t.

When Page began this journey, he had no idea where it would lead. It was a chance stab at a gene clone that marked his first brush with what would become a lifetime study. Today, he travels a more purposeful path in his research. Still, he says, smiling, there is much to be said for happenstance.