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Silence of the Xs

Rcjohnsen rcjohnsen at aol.com
Sun Aug 13 12:34:50 EST 2000

Silence of the Xs
Does junk DNA help women muffle one X chromosome?
SN 158:92-94    Aug. 5, 00

   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
   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
   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.
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 
   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
   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 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. 


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
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
Travis, J. 2000. Human genome work reaches milestone. Science News 158(July

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
10900 Euclid Avenue
Cleveland, OH 44106

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