Huntington Willard, it could be said, is a true X-man. In algebra, X denotes the archetypal unknown quantity. The aptly nicknamed "Hunt" Willard confesses to an inordinate fondness for the unknown. He revels in tackling profound genomic mysteries that confound other researchers and could lead to astonishing new scientific insights--or simply to more mysteries.
The X-Men of comic-book and movie fame find that their superpowers set them at odds with society. Willard has found himself at times disparaged by colleagues for sticking to research paths they believed led only to career-killing dead ends. And, like the X-Men, Willard has assumed dual identities. He is both an active scientist and the activist director of Duke's Institute for Genome Sciences & Policy (IGSP)--the campuswide, multidisciplinary program created to enable Duke to address the broad implications of twenty-first-century genetic advances.
Perhaps most important, X denotes Willard's research on the X chromosome--the sex-determining chromosome that occurs in twos in women, but is paired with a Y sex chromosome in men. After years of studying the way that some genes on the X chromosomes of women are active while others lie dormant, Willard and his colleagues recently reported startling findings. Comparing gene activity on the X chromosomes of forty women, the scientists found unexpected amounts of variation among individuals.
The results have important implications for understanding the differences between men and women in areas such as health and disease. They also offer potential explanations for well-established differences between the sexes. "In essence," Willard says, "there is not one human genome but two--male and female."
The early researchers believed that dosage compensation completely inactivated or "silenced" one or the other X chromosome. That way, all women--and women and men--would have the same dosage levels of encoded genes on their X chromosomes. However, during the 1980s, Willard and his colleagues discovered that some genes on the silenced X chromosomes of women actually remained active. (Male chromosomes are X-chromosomal couch potatoes. They don't practice such dosage compensation. Because they have only one X chromosome, they need all their X-chromosome genes active.)
Despite Willard's early findings that some X genes escape silencing, many scientists still believed that all women had the same patterns of active and silenced genes on their X chromosomes. His most recent research study--co-authored by a former trainee in Willard's lab, Laura Carrel, now an assistant professor of biochemistry and molecular biology at Pennsylvania State University--was published in the March 17 issue of Nature. It compared gene activity on the X chromosomes of forty women. The scientists found surprising variations among the women in the patterns of their genes that were switched on.
The discovery is significant, according to Willard, because "the findings suggest a remarkable and previously unsuspected degree of expression heterogeneity among females in the population," he says. Among other things, this means that women are genetic "mosaics," with any of their cells potentially switching on genes on either of the pair of X-linked genes.
This wide variation among women in X-chromosome gene expression not only points to differences in traits among females, but also between females and males,
Willard says. And an understanding of the genomic differences between the sexes could lead to explanations for the differences in such areas as susceptibility to certain diseases.
"We've always known that the X chromosome was important for disease, and that there are a great many X-chromosome-linked diseases in males," he says. Such rare genetic diseases more likely strike men, because a disease-causing mutation on the lone male X chromosome cannot be compensated for by a protective normal gene on the paired X chromosomes of women. "What fascinates me about these new studies is that they may give us an insight into far more common disorders that show characteristic differences in the frequency between males and females.
"Autism, for example, is about four times more common in males than in females. Why? Rheumatoid arthritis and many other autoimmune disorders are much more common in females than in males. Why? Our results at least raise the possibility that these genes are failing to be fully dosage-compensated, creating a characteristic dosage difference between males and females. And those genes could likely play a role in increasing or lowering the susceptibility of one sex compared with the other to some of these conditions.
"But we also have no idea whether the variation is the same in a fetus in utero or in a newborn, or in a ninety-year-old woman," says Willard. "And it may be that gene expression is changing during that time, and that change may associate with late-onset diseases such as heart disease."
The new findings are only the latest emerging from decades of Willard's research on the phenomenon of dosage compensation. His scientific quest began as a Eureka! moment he had as a Harvard undergraduate. "I was sitting in a library flipping through a journal waiting for a professor who was late. And I came across this paper on X inactivation, and it just struck my fancy. The basic lesson from this paper was, 'we haven't a clue what's going on here.' To me, that was the greatest way to enter a scientific problem, because your imagination can run wild. People were just shrugging their shoulders and saying, 'It makes intuitive sense why males and females would need to equalize dosage of genes.' It was as if a 'miracle' occurs, and it just happens."
Willard recalls that he became instantly fascinated by the scientific mystery of this unknown biological mechanism, which is central to the development of every female. "I must have written every one of my papers as a biology undergraduate on this topic. And I kept reading and writing and exploring different models in my mind." The uncharted machinery of dosage compensation resonated with his (perhaps genetic?) predilection for black-box problems. "I've always been bored by projects where the answer was too obvious--where the answer was going to be one or another known possibilities," he says. "I found it much more interesting to dream about possibilities that just hadn't been described yet."
The fascination endured. When Willard started his own research laboratory after receiving a Ph.D. in human genetics from Yale University in 1979, his first goal was to figure out the machinery the cell uses to shut down X-linked genes during embryonic development. Fifteen years of painstaking work led to the identification of a master genetic switch that turns off such X chromosome genes. But this switch was a peculiar gene, indeed. The huge majority of known genes are blueprints for "messenger RNA" that produces proteins; however, this gene, dubbed XIST, instead produces a type of RNA that controls other genes. These genes that control other genes represent the next great frontier in genomic research, Willard says. Traditionally, geneticists have focused on how genes code for proteins; now they are beginning to explore the "epigenetic" machinery by which genes themselves are controlled.
As he delved into the machinery of X inactivation, he encountered other surprises. The X chromosome control system did not function as a single on-off switch, like the master circuit breaker in a house. Rather, Willard was to discover, it acted more like the multitude of individual electrical switches within that house, with different switches for different genes. He recalls the first inkling he had that X inactivation wasn't an all-or-nothing proposition. "I was teaching an undergraduate class at the University of Toronto back in the late 1980s, and I assigned students what I thought was a simple little project--to look at gene expression on the X chromosome. And the students came up with an answer that made absolutely no sense at that time. They found a gene still being expressed, even though it was on the inactive copy of the chromosome instead of the active copy. And even though I was tempted to simply say, 'You're wrong. It can't happen,' and put a big X across the lab report, we started looking into that question."
At that point, Willard's black box transformed into a treasure chest. He and his colleagues discovered a dozen examples of genes that escaped silencing. In recent years, as the Human Genome Project has yielded the complete structure of the X chromosome, the researchers have used that knowledge to find hundreds more.
They are now exploring not only how the cell decides which genes should escape silencing, but also, why. And they are seeking the origins of the startling variations they discovered among women in the genes that escape silencing. "Maybe the patterns are random, but it's much more intriguing to me to consider that the pattern of this gene activation is inherited," Willard says. "If so, when we compare the X chromosomes of mothers and daughters, or of sisters, or of identical twins, we should see a familial pattern. If it is a pattern in the genome, then we're off on another hunting expedition. Somewhere amidst the vast stretches of DNA on the X chromosome there is some sequence of DNA that tells those genes to be expressed or not expressed. It's another genetic code that we don't understand and can't even begin to articulate."
If the machinery of X inactivation is a fascinating set of nested black boxes, Willard's other major research object, the centromere, has proven a murky Stygian nightmare. The centromere--the point at which paired chromosomes are attached to enable them to navigate through cell divisions--had been largely shunned by scientists, because it was thought to be a genomic wasteland. It seemed to be nothing more than genetic "stuttering"--regions of inanely repetitive DNA code that had no purpose other than to take up space and frustrate biologists.
In fact, the so-called "complete" sequencing of the human genome, announced with great fanfare in 2000, did not include any sequences of the seemingly unfathomable centromeric regions. Willard recalls that, in the 1980s, "there was this series of wonderful papers arguing over whether all this repetitive DNA should be called 'junk,' 'garbage,' or other pejorative terms. But I just sort of took it on faith that nature wouldn't do that. This is 5 percent of the entire genome--a stunning amount to be unimportant and just sitting in a garbage heap at the center of the chromosome."
It was Willard's search to understand the X chromosome that led him to the centromere. He and his colleagues were searching the centromeric region of the X chromosome for DNA sequences that might somehow control X inactivation. He hypothesized that the repetitive elements specific to the X chromosome might act as tags that would key the silencing mechanism. "We did find repetitive sequences specific to the X chromosome centromere," he recalls, "but they had nothing to do with X inactivation whatsoever. So, the experiment didn't work the way we thought it would, but rather than throw it away, it caught my fascination." Willard devoted his efforts to searching for evidence that the repetitive DNA found in the centromeric region was functional rather than just a pile of garbage.
This may mark the point that Willard assumed an X-man persona. "I think that was probably the low point of my reputation in the scientific community," he says, "because people really thought I was crazy. They asked, 'Why are you working on this? It's total junk. You're wasting your time. There can't be a code in there, because it's the same little sequence over and over again. So, by definition, it can't be telling us anything.' I don't know whether I like a challenge or whether I'm pig-headed, but we kept at it."
So, the "pig-headed" Willard and his colleagues invented analytical genetic techniques that enabled them to decipher DNA sequences in the hall-of-mirrors realm of the centromere. One thought was that maybe this repetitive DNA, called alpha satellite DNA, "was just a camouflage that was hiding some other magic sequence that would be buried in there, and we'd have to develop tools that would allow us to get to that magic sequence."
Willard's search for a "magic sequence" was still causing head shaking among colleagues, he says, when his laboratory dropped a scientific bombshell: It announced the creation of a functioning artificial human chromosome. Willard reasoned that if the stuttering DNA was central to the centromere's function in dividing cells, then an artificial chromosome with an artificial centromere containing only "junk" DNA should waltz right along with its natural brethren during the dance of cell division. Sure enough, when Willard and his fellow centromerists synthesized the chromosome and added it to a human cell, they found it worked beautifully. The human cells harboring the artificial chromosomes divided happily ever after, reproducing the artificial chromosomes along with the natural ones.
"Our cells have forty-six chromosomes, and we stuck in a tiny forty-seventh chromosome, and it worked just the way our natural ones do. That was the key breakthrough in that field," he says. "It showed for the first time, definitively, that these alpha satellite sequences confer centromere function."
Willard insists that nature must have evolved these stuttering sequences to mean something important to the cell, since successful cell division is so critical to all life. Still, the nature of that "something" remains unknown. "I'm the first to admit that the fact that our artificial chromosomes work doesn't tell us the code the centromere uses," says Willard. "That just tells us where the code is."
The scientific community reacted to his attempts to build an artificial human chromosome with "a lot of eye-rolling," Willard says. "It was sort of, 'Here he goes again, not giving up on this idea.'" However, he recalls, when he presented his results formally at a 1997 conference in Madrid, "it put to rest the notion of a magic sequence buried in the DNA and established that it was the alpha satellite DNA itself that was being read by the cell." Willard and his colleagues are now tinkering and testing versions of the artificial chromosome, to search for the key characteristics that make the centromere work. The search will be an arduous one, given that the centromeric region comprises some 3-million DNA units on each chromosome.
As a veteran of the scientific centromere wars, Willard deeply appreciates the fact that the science of genomics is full of unknowns--X's--yet to be determined. He has no fear of confronting the unknown. "We now understand only 2 percent of the genome in terms of how it encodes information," he says. "That leaves 98 percent to go."
Unraveling the Human Genome
August 1, 2005