Why, Oh Y?

“By the late 90s, it was clear that the Y chromosome carried more genes than anyone had given it credit for.”

Page has helped clone and sequence the Y chromosomes of humans, rhesus monkeys, mice, and a handful of other species. His group has demonstrated that the human Y carries many genes involved in sperm production, as well as a handful of genes encoding genome regulators that are expressed throughout the body. Along the way, he’s also explored the roots of germ-cell biology in mouse embryonic development, searching for molecular decision makers involved in producing eggs or sperm. Here, Page talks about the race to identify the Y’s sex-determining gene; how his group won that race, but was ultimately wrong about the answer; and where the rest of the Y chromosome has since led him.

Page Sends Out Probes

Rural roots. Page grew up amid farmland on the outskirts of Pennsylvania Dutch country. He loved nature, but he was never really exposed to any scientific research. That all changed when he became the first member of his family to attend college. Swarthmore College was only 90 miles away, but “it was a completely different world,” Page says. He became enthralled with the life of academics, and of scientists in particular. “I came to realize that there were people who spent their time thinking about big ideas and how things worked.” Early in his freshman year, cell biologist Bob Savage drove him to nearby Haverford College for a seminar on the chemical origins of life. “It wasn’t that I was so taken by the subject matter. . . . Just this notion of people traveling around with ideas to share was quite intoxicating for me.”

Basement science. After his junior year, Page spent the summer at the National Institutes of Health (NIH), working on the structure of histones in the basement lab of the late biochemist Robert Simpson, a Swarthmore alum. At night, he bunked in the basement of Simpson’s house. “Talk about an immersion experience in the life of a scientist,” Page says. “I basically became a member of the family for a year.” Page enjoyed the experience so much that he enrolled in only a single seminar the following semester at Swarthmore. He continued living in Simpson’s basement and working at the NIH—and took the train up to Pennsylvania once a week for his class.   

Bookkeeping error. After college, Page joined the Health Sciences and Technology (HST) MD program hosted by both Harvard and MIT. Having worked on chromosomal proteins as an undergrad, he wanted to switch gears and “do something with nucleic acids,” he says. At the advice of MIT’s David Baltimore, another Swarthmore alum, Page connected with David Botstein, also at MIT, and with White’s lab at UMass that first summer when he toothpicked his way to studying sex chromosomes. But figuring out that the phage clone he’d snagged came from the X and the Y took a bit of sleuthing. The variation in the DNA he’d purified from different samples, which came from American Red Cross blood donations, appeared to correlate with sex: in some cases females would have two bands where males and other females had just one, suggesting the snippet of DNA might come from the X chromosome. And there was one band that appeared to come from the Y: it was evident in all of the male samples and in none of the females—except one. “It took me probably six months, but I eventually showed it was a bookkeeping mistake, and that that was actually a male sample that had been mislabeled as female,” Page says.

Page Probes Deeper

Founding fellow. As Page neared the end of his MD program, Botstein mentioned to him that Baltimore was in the process of setting up and staffing the Whitehead Institute. A few months later, Page became the Whitehead’s first fellow; the building opened six weeks after he graduated. “I essentially entered the day it opened, and that was 30 years ago. It’s an altogether unlikely and irreproducible life story, but makes me very much appreciate the role of chance and unlikely opportunities that come one’s way.”

Near miss. “I was scared out of my wits,” Page says about starting his own lab at the Whitehead with no PhD or postdoctoral training under his belt. He decided to continue studying the human Y chromosome, joining many other researchers in the search for a gene that set male development into motion in the mammalian embryo, which defaults to a female anatomy. “There was a very fierce international competition to chase down the sex-determining gene on the Y chromosome,” Page recalls. At one point, he and his group thought they’d struck gold with a gene dubbed ZFY, which encodes the zinc finger Y-chromosomal protein. “In late 1987 we published, to enormous fanfare, what we thought was the sex-determining gene on the Y chromosome. It turned out to be a miss, a near miss;  [ZFY] was the gene next door to SRY.”

Mapping the Y. A few years later, after SRY was correctly identified as the sex-determining gene on the Y by the U.K.’s Peter Goodfellow and Robin Lovell-Badge, molecular biologists began interrogating SRY or left the field altogether, Page says. “People went home, found other jobs. . . . Because there was the assumption that there was nothing else there on the Y, that was it.” But Page shifted his focus from the short arm of the chromosome, where SRY resides, to the long arm. Because the Y chromosome doesn’t cross over with the X, it doesn’t lend itself to traditional genetic linkage mapping—part of the difficulty the field faced in identifying the sex-determining gene. Instead, Page turned to naturally occurring deletions in the chromosome. Within a few years, he and others began identifying deletions on the long arm that caused spermatogenic failure and resulted in infertility. “By the late 90s, it was clear that the Y chromosome carried more genes than anyone had given it credit for,” Page says.

Hall of mirrors. With the advent of PCR, the team continued to refine its maps, and in 1992, Page and his colleagues succeeded in cloning the entire Y, giving them a near-complete physical map of the chromosome. Still, not all of Page’s questions about the Y’s structure were answered. “It was extremely confusing at first, because it turned out that there were big chunks of DNA that were present multiple times on the long arm of the Y chromosome,” Page says. He teamed up with colleagues at Washington University in St. Louis to sequence the human Y. The task would require the invention of a new sequencing technique, dubbed SHIMS (single-haplotype iterative mapping and sequencing), that allowed for much longer reads of 150,000 to 200,000 base pairs. Page finally identified the source of his confusion: the Y chromosome’s long arm, some 42 million bases long, carried a handful of large palindromes, the largest of which spans three million bases of DNA. His team would go on to show that these Y palindromes make the chromosome susceptible to the deletions that cause spermatogenic failure. In 2012, the team found that one particularly harmful deletion on the long arm of the Y crops up anew in one in every 2,400 newborn boys. “This is a spectacularly high rate of new mutation,” Page says. “We began to realize that this seemingly chaotic hall of mirrors actually was like a crystal palace.”

The evolving Y. As Page’s group generated more-detailed maps of the Y, the researchers began to suspect that the chromosome, along with the X, had evolved from what were once ordinary autosomes, an idea originally proposed almost a century earlier. One of the first clues came in the mid-90s when Page’s team discovered that a family of genes—dubbed DAZ for “deleted in azoospermia”—had come to the Y by a transposition of an autosomal segment. Over the next few years, the researchers began to uncover pairs of genes that existed in copies on both the X and Y chromosomes and appeared to have been retained from the ancestral autosomal pair, serving as “living fossils” that detailed the differentiation of the sex chromosomes. In 1999, Page and his then graduate student Bruce Lahn published an analysis showing that the X and Y had diverged in four discrete steps, starting some 200 million to 300 million years ago, as bits of the chromosomes lost their ability to cross over. “I would say that paper from 1999 was, for me, and I think for the field, the turning point in recognizing that the X and Y chromosomes had evolved from ordinary autosomes. . . . We now had the smoking gun at a molecular level.” (See “Doris Bachtrog: Sex Chromosome Wrangler.”)

Palindrome problems. One particular Y chromosome variant that Page’s group studied is what’s known as an isodicentric Y or an iso-Y. “It’s an anomaly of the Y chromosome in which the entirety of the chromosome becomes a palindrome,” Page explains. His group discovered that these chromosomes resulted from aberrant crossing over between two copies of the Y (after the chromosome has replicated during cell division), if the two copies were aligned head-to-toe. Page was struck by the wide range of phenotypes displayed by the people carrying these iso-Y chromosomes: men who were infertile but otherwise healthy; people with ambiguous external genitalia; people who had clearly developed as females, but nonetheless carried iso-Ys in at least some of their blood cells. Page’s group reasoned that, because iso-Y chromosomes have two centromeres, they were prone to being lost during cell division. And if the iso-Y is lost, what would remain is an XO genotype—typically associated with Turner syndrome, a developmental disease that afflicts girls. Sure enough, when Page and his colleagues took a closer look at the individuals carrying iso-Ys, “in fact, there were a number of girls and women who had been diagnosed with Turner syndrome but who were carrying this isodicentric Y in some of their cells,” Page says. “They were actually mosaics in many cases.”

Page Overturns

Germ cell meiosis. Early on in his research into the sex chromosomes, Page realized that he ought to also study germ-cell biology. Massive differences in male and female gamete production can be traced back to embryonic development and the initiation of meiosis in the primordial germ cells. In females, these cells initiate meiosis and begin to develop into eggs within just a few days of arriving in the developing ovary. In males, the germ cells reach the developing testis and simply “hunker down” until it’s time for sperm production to begin in puberty, Page explains. These differences in the timing of meiotic initiation led to the hypothesis that this is what determines whether a germ cell becomes an egg or a sperm. But in a 2013 Nature Genetics paper, Page and colleagues found that even in the absence of meiosis, mouse ovarian germ cells became oocytes that could be ovulated, fertilized by sperm, and even develop into two-cell embryos. “This pretty much blew up the idea that fetal initiation of meiosis represented commitment to the oocyte-like development,” Page says.

Sex chromosomes and disease. Page, who never liked the idea that the Y chromosome was headed for extinction, has spent much time defending Y’s honor, he says. While the idea of the disappearing Y chromosome has, by now, largely been “laid to rest,” he says, “I think that debate has kept us from really considering more broadly the role of the X and Y chromosomes in sexual dimorphism.” Specifically, his recent work has led Page to recognize the vast differences between human males and females in the incidence and severity of disease. Autoimmune diseases such as rheumatoid arthritis and lupus, for example, are more common among women, while autism spectrum disorders are more prevalent in boys. “These sex biases in incidence and severity are not the exception; they’re actually the rule,” Page says. “There’s no simple explanation in our anatomies as to why these differences should exist, and I think that ultimately these differences will trace their biological origins back to the X and Y chromosomes.”

The roles widen. In a paper published last year, Page and his colleagues compared the Y chromosomes of eight mammals, including the human, other primates, and the mouse, along with the chicken Z, identifying surviving gene pairs that existed in copies on both the X and the Y. On the human Y, they found 12 such surviving genes, and all were global regulators of gene expression, such as chromatin modifiers. “These are powerful, influential players in the lives of our cells,” Page says. What’s more, the group learned, both the X and Y copies of these genes, which are not identical, are widely expressed throughout the body, and a cell’s sex chromosome genotype determined the isoforms it expressed. “We begin to consider the possibility . . . that some fundamental molecular difference between XX and XY cells could potentially contribute to these very different susceptibilities to a wide range of diseases outside the reproductive tract.”

Greatest Hits

• Identified deletions on the long arm of the Y chromosome that caused the failure of spermatogenesis and resulting infertility.  
• Cloned the human Y, making it one of the first two cloned human chromosomes.
• With graduate student Bruce Lahn, helped detail the evolution of the sex chromosomes from a pair of ordinary autosomes.  
• With colleagues at Washington University in St. Louis, developed a new sequencing technique, dubbed SHIMS (single-haplotype iterative mapping and sequencing), that allowed them to sequence the human Y chromosome, leading to the identification of large palindromes that render the chromosome susceptible to infertility-causing deletions.  
• Overturned the idea that differences in the timing of meiotic initiation determined whether a germ cell becomes an egg or a sperm, demonstrating that mouse ovarian germ cells can become oocytes even in the absence of meiosis.  
• In 2014, identified 12 surviving gene pairs that exist in different copies on the X and the Y chromosomes that may explain sex differences in disease susceptibility.