Silence of the Xs

Here’s a riddle that will stump most people. What do calico cats—those popular felines with a patchwork of orange-red, black, and white fur—have in common with women who lack sweat glands on portions of their bodies? A clue: Calico cats are almost always female.

The women and cats illustrate the remarkable fact that all female mammals are actually mosaics of cells with two different pedigrees. Early in embryonic life, when they’re merely balls of cells, female mammals silence one of their two X chromosomes within each cell. Each of the cells randomly decides which X—the one inherited from the mother or the one from the father—it will inactivate. As these embryonic cells replicate, their descendants in the adult animal retain the chromosomal choice that the original cells made.

In the case of a calico cat, the feline’s parents passed on different versions of X chromosome genes related to coat color. As for the women with only a partial supply of sweat glands, one of their two X chromosomes must have a gene mutation that prevents patches of skin with the mutation from making the glands.

The reason female mammals bother inactivating an X chromosome is self-preservation. Over the course of evolution, the mammalian Y chromosome has degenerated so much that it now shares few genes with its more robust counterpart, the X chromosome. That means that women, with their double dose of X chromosomes, have a genetic surplus compared with men.

To ensure that the sexes work with similar doses of X genes, which scientists believe is critical for development, female mammals evolved the ability to muffle one of their sex chromosomes. (In contrast, male fruit flies double the activity of their single X chromosome.)

Scientists first recognized the phenomenon of X inactivation 4 decades ago. Ever since, they’ve sought to understand how the silencing of almost an entire chromosome occurs. The situation is anything but simple. In one recent study, for example, biologists found that many more X chromosome genes than expected escape inactivation.

Another new study offers an intriguing explanation for that finding and suggests a mechanism for how X inactivation occurs. Supporting a theory proposed 2 years ago, a research team has uncovered evidence that DNA sequences usually dismissed as junk DNA without any function actually help determine what genes on the X chromosome become suppressed.

The new work doesn’t come close to resolving all the questions surrounding X inactivation, but optimistic investigators contend that they’re closing in on a better understanding of the puzzling event.

“At the moment, because we don’t known how to put together the disparate bits of information into a coherent picture, it’s looking complex. Ultimately, it’ll look a lot more simple and elegant,” says Neil Brockdorff, who studies X inactivation at the Imperial College School of Medicine and Hammersmith Hospital, both in London.

Feline brains

What led scientists to the process of X chromosome inactivation in the first place were some cats, though perhaps not calicos. In 1948, while studying the brains of felines, Murray L. Barr and his graduate student E.G. Bertram noticed dark, drumstick-shaped masses inside the nuclei of nerve cells. The pair also discerned a puzzling pattern: The masses appeared in the cells of female cats but not in those of males—an observation that eventually led to a test that Olympics officials have used to ferret out men competing as women.

The dark masses, now known as Barr bodies, were pieces of chromatin—the amalgamation of DNA and protein that makes up chromosomes. In 1959, Japanese cell biologist Susumo Ohno announced that each Barr body was an X chromosome, albeit a highly compacted one.

That same year, a second research team created mice with just a single X chromosome and found that the animals were healthy and fertile. This finding suggested that a second X chromosome might not be necessary at all.

Building on those data as well as her own work on mice with mottled coats, Mary F. Lyon of the Medical Research Council’s Mammalian Genetics Unit in Harwell, England, proposed in 1960 the idea of X inactivation, with the shutoff chromosome becoming the Barr body. While some critics challenged Lyon’s hypothesis, it quickly garnered enough support that X inactivation was called—and still is, by some scientists—Lyonization.

Even in those early days, however, scientists recognized that this unusual chromosomal silencing wasn’t absolute. Lyon suggested, for example, that females wouldn’t inactivate an X chromosome gene if it had a counterpart on the Y chromosome. In such a case, both sexes still would have two active copies of the gene in question.

Further evidence that some X genes avoid inactivation comes from men and women born with an extra X chromosome. A remarkable wrinkle of the inactivation phenomenon is that cells appear to count X chromosomes. Even if there are more than two X chromosomes, the cells shut down all but one. Still, XXY men are sterile, and XXX women have other health problems, implying that some genes on those additional X chromosomes remain active.

Genes that escape

Over the years, scientists indeed have identified a handful of genes that escape X inactivation. Hoping to tally a more comprehensive list, Huntington F. Willard of Case Western Reserve University School of Medicine in Cleveland and his colleagues recently analyzed the activity of more than 200 genes mapped to the X chromosome, about 10 percent of the chromosome’s estimated total.

They fused cells from a woman with those of a female mouse, creating hybrid cells whose descendants in a cell culture gradually lose the human chromosomes. During this process, the scientists were able to procure hybrids containing either the normal human X chromosome or its inactivated counterpart. By comparing genetic activity of the two kinds of cells, say the researchers, they can determine which genes from the inactivated X normally remain on.

In the Dec. 7, 1999 Proceedings of the National Academy of Sciences, Willard’s team reported that an unexpectedly large number of X chromosome genes, 34 of 224, resist silencing. Moreover, most of these, called escapees, don’t have a partner on the Y chromosome. “Strict dosage compensation of all genes on the chromosome isn’t necessary,” concludes study coauthor Laura Carrel.

Brockdorff agrees. He suggests that only the subset of X chromosome genes involved in embryogenesis demands dosage balancing between the sexes. Just as cells usually survive a recessive mutation—a mutation that disables only one of a cell’s two copies of a gene—they can manage having double the amount of the proteins encoded by many X chromosome genes, he says.

Another finding that emerged from the census of genes that avoid inactivation reflects the evolutionary history of the X chromosome. Of the 34 escapees, 31 reside on the short arm of the chromosome, Carrel and her colleagues report.

That disparity doesn’t surprise biologists. Previous research had indicated that the long arm derives from an early evolutionary form of the X chromosome and that the short arm was added at some point after marsupials diverged from mammals that form a placenta to support a fetus. The large number of escapees on the short arm, says Lyon, may reflect the fact that X inactivation hasn’t yet fully taken hold in that relatively new stretch of DNA.

Quantifying the degree of escapism from X inactivation is complex since gene silencing isn’t always an all-or-nothing affair. Scientists have found that several genes on the inactivated X remain active but at a much lower level than their counterparts on the active X chromosome.

Noting that the study by Carrel’s team didn’t gauge the degree of a gene’s activity, Brockdorff suspects that many of the genes labeled as escapees actually experience partial suppression.

Stifled genes

While X inactivation in people isn’t as comprehensive as scientists first thought, it still stifles the large majority of the more than 2,000 estimated genes on the chromosome. If a recent theory proves true, this efficiency may depend on boosts from DNA long thought to have no useful function within mammals.

The mechanism by which chromosomal silencing comes about remained a complete mystery for several decades after Lyon’s proposal. Finally, in the early 1990s, scientists studying X inactivation identified a key gene, called Xist (pronounced “exist”). As usually happens in gene expression, cells that read this gene create a long piece of ribonucleic acid (RNA), a singlestranded chemical relative of DNA. But rather than translating the information encoded in the RNA to build a protein, which is the usual next step in gene expression, the Xist RNA remains untranslated. The RNA is the final product.

These bits of RNA have a thing for X chromosomes. At the appropriate time in development, they appear to coat the entire length of one of the X chromosomes, which then condenses into a Barr body. In fact, scientists have added Xist to other chromosomes and inactivated expanses of them (SN: 3/22/97, p. 173), though never as extensively or efficiently as with X chromosomes.

There’s additional evidence that something about the X chromosome makes it especially prone to inactivation. On rare occasions, people are born with fragments of an X chromosome latched onto another chromosome. Sometimes, that other chromosome experiences inactivation, but not as thoroughly as the X does.

Seeking to explain such observations, Stan M. Gartler and Arthur D. Riggs in 1983 theorized that the X, compared with other chromosomes, is enriched in some DNA sequence that facilitates gene suppression. The two called this putative piece of DNA a “way station” or “booster element” because it would help inactivation spread across the X chromosome.

That’s where junk DNA enters the story. In 1998, Lyon put forward DNA sequences called LINE-1 elements, or L1s, as candidates for the boosters discussed by Gartler and Riggs.

Most of the human genome—some estimates suggest as much as 97 percent—actually consists of apparent junk DNA sequences such as L1 that have accumulated during millions of years of mammalian evolution. They don’t serve any obvious function other than to make more copies of themselves. “The model is that most of these things, because they’re selfish, basically propagate and go where they can,” explains Evan Eichler of Case Western Reserve University Medical Center.

In her 1998 proposal, Lyon noted that a few studies indicated that the X chromosome has a greater abundance of L1s than any nonsex chromosome and that interactions between Xist RNA and L1 elements could conceivably facilitate gene silencing by helping this RNA spread along a chromosome.

The studies that Lyon cited to support her hypothesis offered relatively crude estimates of the amount of L1 elements in various chromosomes. That’s why Eichler and his colleagues recently performed a more exhaustive analysis, drawing upon the growing availability of DNA-sequence data. More than 26 percent of a person’s X chromosome consists of L1s, about twice the proportion for other chromosomes, Eichler, Carrel, Jeffrey A. Bailey, and Aravinda Chakravarti—all of Case Western—report in the June 6 Proceedings of the National Academy of Sciences.

The greatest concentration of L1 sequences occurs right around Xist and accounts for almost 40 percent of the stretches of DNA adjacent to the gene. This feature matches another property Gartler and Riggs predicted their booster elements would have.

Eichler’s team compared regions on the X where genes normally avoid silencing with spans that become inactivated. “It was surprising how clear the data are. Regions that were subject to X inactivation were really enriched in L1 elements. Regions that escape inactivation . . . had a lower L1 content than autosomes [chromosomes other than X or Y],” Eichler says.

Particularly telling was that only certain classes of L1s were more abundant on the X chromosome. Investigators gauge the age of L1s by their differing DNA sequences. Eichler’s team discovered that only the younger classes of L1s, those that had made copies of themselves within the past 60 to 100 million years, have a greater-than-normal presence on the human X chromosome.

The proliferation of these L1s in relatively recent times may explain why X inactivation is more stable and complete in placental mammals than in marsupials. The two lines of animals diverged from a common ancestor before this presumed burst of L1 replication on the X chromosome.

That would leave marsupials with fewer L1s, thwarting inactivation.

Anthony V. Furano of the National Institute of Diabetes, Digestive and Kidney Diseases in Bethesda, Md., praises the analysis by Eichler’s team. Yet he remains skeptical that L1s help Xist RNA spread and smother genes. Since the inactivated X chromosome gets copied later than other chromosomes when a cell divides, L1s may simply have an increased chance of landing on that particular chromosome, notes Furano.

“Nobody really knows why L1 goes where it goes,” he contends. “The LINEs may just be a red herring, a symptom of something else going on with X. It may be a consequence of X inactivation rather than a cause.”

To firm up the case, Eichler’s group will look to the distribution of L1s in the mice, animals in which X inactivation happens more completely than in women. Eichler predicts that the mouse X chromosome will also have an unusually high abundance of L1s, particularly around genes that escape inactivation on the human X but become suppressed on the mouse chromosome.

“To actually think of a good experiment that would directly test [Lyon’s L1] hypothesis is not that straightforward,” notes Brockdorff. One experiment that scientists are considering would require building two artificial chromosomes, each containing Xist, that differ only in their abundance of L1s.

All the scientists studying X inactivation stress that there must be more to how Xist RNA shuts down a chromosome than the mere presence or absence of L1s. They continue to look, for example, for proteins that bind to that RNA.

Still, Eichler’s latest work has given him a greater appreciation for the possibility that DNA sequences such as L1 elements have some role in human biology. “We as scientists have this preconceived notion, based on very little data, that this selfish DNA is nothing more than junk.

I think junk is a very unfortunate term. It’s more a reflection of our ignorance,” he says.