Silence of the Xs
Does junk DNA help women muffle one X chromosome?
SN 158:92-94 Aug. 5, 00
By JOHN TRAVIS
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.
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
Green circles and blue diamonds show the location of genes on the X chromosome
that escape inactivation. Most are on the chromosome's short arm.
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 Union
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—Lionization.
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.
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.
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 l990s, 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 (F<NA), a
single-stranded 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.
One image (left) of the nucleus in a woman's skin cell shows both X
chromosomes labeled with a fluorescent marker (red). In the other image, a
different marker (green) identifies the inactivated X by highlighting an RNA
strand from the gene Xist.
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: 3/22197, 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 Lls, 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 Ll 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 Lls 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 Lls, 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 Ll elements. Regions that escape inactivation . . . had a lower Ll content
than autosomes [chromosomes other than X or Y]," Eichler says.
Particularly telling was that only certain classes of Lls were more abundant
on the X chromosome. Investigators gauge the age of Lls by their differing
DNA sequences. Eichler's team discovered that only the younger classes of Lls,
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 Lls 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 Ll replication on the X chromosome. That would leave
marsupials with fewer Lls, 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 Lls help Xist RNA spread and smother genes. Since the
inactivated X chromosome gets copied later than other chromosomes when a cell
divides, Lls may simply have an increased chance of landing on that
particular chromosome, notes Furano.
"Nobody really knows why Ll goes where it goes," he contends. "The LlNEs
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 Lls
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 Lls, 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 Ll]
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 Lls.
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
Lls. 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 Ll elements have some role in human
biology. "We as scientists have this preconceived notion, based on very
little data, that this selfish DNA's nothing more than junk. I think junk is
a very unfortunate term. It's more a reflection of our ignorance," he says.
References:
Bailey, J.A. . . . and E.E. Eichler. 2000. Molecular evidence for a
relationship between LINE-1 elements and X chromosome inactivation: The Lyon
repeat hypothesis. Proceedings of the National Academy of Sciences 97(June
6):6634.
Carrel, L. . . . and H.F. Willard. 1999. A first-generation X-inactivation
profile of the human X chromosome. Proceedings of the National Academy of
Sciences 96(Dec. 7):14440.
Disteche, C.M. 1999. Escapees on the X chromosome. Proceedings of the National
Academy of Sciences 96(Dec. 7):14180.
Lyon, M.F. 2000. LINE-1 elements and X chromosome inactivation: A function for
"junk" DNA?. Proceedings of the National Academy of Sciences 97(June 6):6248.
Further Readings:
Brockdorff, N. 1998. The role of Xist in X-inactivation. Current Opinion in
Genetics & Development 8:328.
A demonstration of X-inactivation is available at
http://wsrv.clas.virginia.edu/~rjh9u/xinactanim.html.
Travis, J. 2000. Human genome work reaches milestone. Science News 158(July
1):4.
Sources:
Neil Brockdorff
X-inactivation Group
MRC Clinical Sciences Centre
Imperial College School of Medicine
Hammersmith Hospital
DuCane Road
London W12 0NN
United Kingdom
Laura Carrel
Department of Genetics, BRB 701
Case Western Reserve University School of Medicine
2109 Adelbert Road
Cleveland, OH 44106-4955
Evan E. Eichler
Department of Genetics
Case Western Reserve University
BRB720
10900 Euclid Avenue
Cleveland, OH 44106
