New genetics of the inactive X chromosome reveals its surprisingly active role in the cell
The sex chromosome has been misunderstood for nearly sixty years. Researchers at the Whitehead Institute are working to restore its reputation.
Courtesy of the National Human Genome Research Institute
Courtesy of Dr. David Page
Imagine you are back in high school biology. It is the first day of the genetics unit, and your teacher projects an image of various colorful worms of different shapes and sizes on the whiteboard. These neon-striped figures aren’t worms, though; they are tightly wound strands of DNA — all of our DNA.
You’ve just been introduced to the human chromosomes: 46 strands of DNA, packed into each of the 30 trillion cells in our bodies, that comprise the body’s entire blueprint. Pointing at the picture, your teacher tells you that our chromosomes exist in twenty-three pairs of identical copies. For the most part.
The one exception is our twenty-third pair. This duo comprises the sex chromosomes, which determine our biological gender at conception: an X and Y chromosome in typical males and two X chromosomes in typical females.
The X and Y chromosomes could not be more different from one another. Bearing nearly one thousand genes, the X chromosome stands several times taller than its Y counterpart, whose gene count is in the dozens. In a male’s complete set of chromosomes, the X and Y stick out like sore thumbs compared to the twenty-two identical twins that make up the autosomes, or the non-sex chromosomes. On the other hand, the two X chromosomes in females look identical, just like the autosomes. But there’s indeed a vital difference between the two.
Nearly sixty years ago, English geneticist Mary Lyon discovered that one of the two X chromosomes in every female cell is genetically silenced in a process known as X inactivation. Lyon argued that this balances the number of X-linked genes expressed between XX females and XY males, a conclusion long taught in numerous biology textbooks. However, recent work suggests that it’s wrong to think the allegedly inactive X chromosome is doing nothing. Rather, it may be a key contributor to sex differences in biology.
Sexes of most species are inherently different in how they look and experience life. In humans especially, there is a wide variety of sex-based differences in health and disease that scientists have yet to understand in full. Many diseases and disorders, including autism, systemic lupus, and several cancers, vary in prevalence and severity between males and females. What happens on the cellular level to induce these differences has been a long-standing question in science — one that geneticist Dr. David C. Page has been chasing for decades. New research from his lab at the Whitehead Institute for Biomedical Research reveals unexpected functions of the inactive X chromosome, “Xi” for short, opening new insights into the fundamental differences between the sexes.
The real sex chromosomes
Excluding environmental and social determinants, all biologically-based differences between XX and XY individuals find their origins in the sex chromosomes. While this might seem obvious, it isn’t a reality that the field acknowledges, says Page. Frankly, when it comes to research on sex differences in health and disease, the sex chromosomes aren’t at all in the spotlight. But in the Page Lab, they are.
Since the lab was first established at the Whitehead Institute in 1984, its research has defended the honor of the tiny Y chromosome, which has historically been neglected by scientists for its seemingly insignificant number of genes compared to its chromosomal peers. In 1990, the decades-long search for the sex-determining gene was resolved when Dr. Andrew Sinclair and his team identified a Y chromosome gene called SRY.
In February 2023, research published by Page’s group in Cell Genomics revealed that the active X chromosome — the most intensely studied chromosome in human genetics — is virtually identical between male and female cells. When Page shared this finding with members of the National Academy of Medicine at their 2023 Annual Meeting, he was met with many puzzled looks: “Isn’t the X a female chromosome?”
“No, there is nothing female about the first X chromosome,” he responded. “It might as well be the 45th autosome.”
If the active X in females and the single X in males are the same, then the search for what drives sex-based differences in humans must look elsewhere. Work from the Page group suggests that only Xi and Y are responsible for assigning our sex at birth — that they are the real sex chromosomes. “We can stop saying that females are XX and males are XY,” Page asserted, “and start saying females are Xi and males are Y.”
The inactive X is not so inactive
The second X chromosome has carried the burden of being called “inactive” for sixty years. “That does not invite people to devote their careers to studying it,” said Page. Current work on Xi focuses on the mechanism underlying its inactivation and not much else. But around ten years ago, the Page Lab unintentionally embarked on one of the first journeys in Xi research beyond its inactivation.
The group began studying naturally occurring cases of sex aneuploidy — a genetic disorder defined by an abnormal number of sex chromosomes — to better understand X and Y’s impact on human health. While most of us are either XY and XX, some people can be born — and live, albeit with severe health consequences — with one, two, or even three extra copies of either X or Y. By quantifying gene expression in cells with widespread variety in sex aneuploidy, Page and his colleagues could trace how “adding” one X or Y chromosome at a time influences cellular activity. In cooperation with the National Institutes of Health and several other clinicians, the Page Lab received skin biopsies and blood samples from 176 people with different combinations of X and Y chromosomes.
“We didn’t know we’d end up studying Xi,” Page recalled. But the best science, he said, arises when something unanticipated appears in your peripheral vision. “You turn your gaze towards it and, before you know it, you’re walking in a different direction.”
While the study was not designed to explore Xi, X inactivation inevitably made it about Xi. As Mary Lyon argued, nature permits only one active copy of X in every cell, possibly to balance X-linked gene dosage for survival. For people with more than one copy of X, all but one is inactivated in the womb, which means that every person with extra copies of X really has extra copies of Xi. And by comparing gene expression across aneuploidies of increasing X, “we were going to get a quantitative description of the Xi like there’s never been,” Page noted.
To the researchers’ surprise, they observed clear changes in the expression of thousands of genes with each addition of Xi. “So many genes throughout the genome seem to care about how many X chromosomes you have,” said Dr. Adrianna San Roman, a postdoc in the Page Lab and first author of both studies. These affected genes were distributed throughout the human genome — some were on the active X chromosome, and many were found on the autosomes. For Xi, this pointed to a new and robust description of its purpose beyond inactivation: gene regulation. Targeting a wide spread of genes, Xi may play a crucial role in the life of a cell. It seems that the inactive X chromosome has been misnamed.
Page is no stranger to a misunderstood chromosome. As his group began to unravel the mysteries of the inactive X decades after doing the same for the Y, Page thought, “Oh my gosh, the human Xi is as misunderstood as the Y chromosome ever was.”
The impact of Xi “dose” on autosomal and the active X gene expression may be the genetic basis of the symptoms of multiple-X aneuploidies. At the same time, an important question is raised about what the inactive X is doing in people without sex aneuploidy: could it be a driver of sex differences in typical males and females? In other words, do Xi and Y impact autosomal and active X gene expression differently?
To explore this, the researchers in Page’s group compared their results from investigating multiple-X and multiple-Y aneuploidies. Again, they saw something surprising: for every additional Xi or Y chromosome in an individual, many autosomal genes responded in strikingly similar ways. They published the results in a second Cell Genomics paper in January 2024. “The correlations that we see are remarkable,” said San Roman, “I was very confused about this at first.”
Since having either Xi or Y is the only cell-intrinsic way in which the human sexes are different, it was easy to expect that having increased numbers of one or the other would impact gene expression differently. “But that’s not what the data was telling us,” said Page.
These unexpected findings may reflect the similar evolutionary origins of X and Y. The two were descendants of an ordinary pair of autosomes, which some believe could have contained an identical set of genes that were crucial for cell survival and thus preserved over time. These hypothetical gene pairs could explain Xi and Y’s similar effects on gene expression observed in the Page group’s findings. But while this theory may explain the shared functions of Xi and Y, the question remains: where do sex differences come from?
San Roman notes one hypothesis. It stems from an observation from the sex aneuploidy study: while Xi and Y influence autosomal gene expression quite similarly, it seems that Xi can push a little harder. Genes that responded — either increasing or decreasing in expression — to the addition of Xi seemed to do so with slightly larger effect sizes, San Roman says. Perhaps some of the sex biases we see in health and disease are simply due to differences in how much Xi and Y influence gene expression.
For Xi research, this is just the beginning
The findings from the Page Lab raise several questions about Xi’s function. For example, by what mechanism is Xi exerting autosomal gene regulation? One possibility may involve gene products called non-coding RNAs expressed exclusively on Xi. One of these is XIST, the primary driver of X inactivation. Dr. Phil Sharp, a 1993 Nobel Laureate and molecular biologist at the Koch Institute for Integrative Cancer Research, has done significant work in non-coding RNAs. He suspects that those such as XIST could underlie Xi-mediated gene regulation.
“It’s mechanistically interesting,” said Sharp. He hypothesizes that higher-fold dosages of XIST could form condensates in multiple-X aneuploidies. A contemporary principle in biology, membrane-less RNA-rich condensates have been demonstrated to influence cell activity in various ways.
Other gene products, such as proteins, are being investigated to understand Xi’s role in the cell. Dr. Kathy Liu, a biochemist from the University of Pennsylvania Perelman School of Medicine, is also interested in the biology of sex-based differences; she studies homologous proteins of the X and Y chromosomes in the context of human cancers. For her, the Page group uncovering new genetics of Xi is “really paradigm-shifting,” but it demands plenty more research. Gene expression doesn’t necessarily equate to protein function, she said. “Without protein-level information, I’m not one hundred percent convinced.”
Much of the inactive X chromosome remains a mystery, but the Page Lab has taken one of the first of many steps toward resolving its overlooked role in the cell. It will likely be a long time before it’s fully clear how Xi contributes to sex differences in health and disease, but a sense of familiarity keeps Page hopeful. “I know this gig,” he said. “You take a chromosome that is underappreciated, and you study it like crazy. And people will not believe you until you’re almost done.”