03.2020 | THE SCIENTIST 25F
or a person with two X chromosomes, full expression of the
genes on both could mean a disastrous double dose of their pro-
tein products, interfering with the delicate balance of protein
expression and interactions all over the body. But cells have a simple
solution: turn off one X chromosome and crumple the extra genes
into a quieted mass of DNA called a Barr body. A long noncoding
RNA known as XIST (pronounced “exist”), which is expressed from
the “inactive” X itself, plays a key role in this process. Acting only on
the chromosome it’s transcribed from, XIST coats the DNA, turning
it into silent heterochromatin.
Except there’s a problem. Certain genes just won’t stay mum.
Scientists had long known that some genes on X chromosomes
aren’t subject to silencing, but the list of these “escapees” has grown
over the past two decades. Somehow, about a quarter of the approxi-
mately 1,150 genes on the purportedly silenced human X speak out:
they avoid coating by XIST. Some of these escapees are expressed just
a bit, others quite a lot. Some appear to be beneficial, possibly help-
ing to protect XX individuals from diseases such as cancer. Other X
genes cause damage when expressed from both chromosomes, pos-
sibly predisposing women to autoimmune disorders such as mul-
tiple sclerosis or lupus.
Recent clues suggest that the signals to activate a Barr body gene
lie in the DNA sequence and proteins that attach to it. Some Barr
body genes, including XIST itself, actively control the silencing of
other stretches of the X. Such research into the nature of X escape
has major implications for understanding biological differences
between the sexes, says Sundeep Kalantry, a geneticist at the Uni-
versity of Michigan Medical School. Scientists have long pointed to
sex hormones and genes on the Y chromosome as exclusive explana-
tions for those differences. “I think this is being questioned by many
groups,” Kalantry says. “The concept is out there that genes that
escape X inactivation are influencing female-specific physiology.”Identifying the escapees
English geneticist Mary Lyon, who originally proposed
X-chromo some inactivation in 1961^1 while working at the UK’s
Medical Research Council in Harwell, suspected early on that
certain genes wouldn’t be shut off.^2 At least 29 genes reside in
the two pseudoautosomal regions (PAR1 and PAR2) shared by
the X and the Y chromosomes. These regions at the tips of the
sex chromosomes typically stay active even in the Barr body, so
that XX and XY individuals get the same doses of those genes.
Studies on non-PAR escapees began to trickle out in the 1970s^3
and ’80s,^4 but the extent of the phenomenon wasn’t known, and low
expression of these escapees was presumed to have a minimal effect
on biology. Only in the past 15 years have researchers begun to appre-
ciate the importance of escape, in terms of the number of genes, their
significant expression levels, and their physiological relevance. Genes
on the inactivated X rarely reach 100 percent of active-chromosome
expression levels; “escape” is usually defined as achieving RNA lev-
els of at least 10 percent of the expression seen in the active X. But it
turns out that even a small amount of expression from the Barr body
can make a biological difference.The first real catalog of escapees, published in 2005,^5 was aston-
ishing for its size, recalls Carolyn Brown, a molecular geneticist at the
University of British Columbia in Vancouver who was not involved
in the project. Researchers from the Penn State College of Medicine
and Duke University forced active and inactive human X chromo-
somes into rodent cells, creating a panel of hybrid cell lines. Then they
looked for mRNAs derived from those human Xs. Ignoring the PARs,
these researchers found that while 458 of the genes they measured
were silenced on the inactive X in most or all of the cell cultures, 154
others—a whopping 25 percent—were expressed in at least some cells.
(This would later turn out to be a much higher rate than in mouse
X chromosomes, in which 3–5 percent of Barr body genes escape.^6 )
The diversity of escapees was also readily apparent, with genes
for mitochondrial enzymes and blood cell surface proteins among the
many on the list. Many escaped genes are transcriptional regulators,
says Brown, giving them ample opportunity to influence biology.
In a 2017 survey of escapees in human tissues,^7 computational
biologist Ta r u Tukiainen, then a postdoc at Massachusetts General
Hospital and the Broad Institute, confirmed the escape rates seen in
rodent-human hybrid cells and described remarkable variability in
expression levels of individual escapees.
In an analysis of transcriptomes from 940 individual blood cells
from four different women, she assessed the activity of 165 X-linked
genes—those expressed at high enough levels to be identified by single-
cell RNA sequencing—including several PA R genes. She found that
only 129 were reliably silenced. Others were silenced only in some of
the women, or in some of their cells.For example, the PAR1 gene ZBED1 was expressed by the inac-
tive X in all four women, making up 20–50 percent of total ZBED1
mRNAs. The known escapee MSL3, which encodes a nuclear protein
thought to be involved in chromatin organization and regulation of
transcription, was expressed equally from both X chromosomes in
one woman, but transcripts from the inactive X only made up 10–20
percent of MSL3 RNAs in two other women.
To interrogate whether escape might contribute to sex differences,
Tukiainen analyzed transcriptomes from men and women who partic-
ipated in the National Institutes of Health’s Genotype-Tissue Expres-
sion (GTEx) Project. Her dataset covered more than 5,500 tran-
scriptomes of 29 different tissues from 449 people. Of the 82 known
escapee genes she analyzed, 74 percent showed considerable differ-
ences in expression between men and women in at least one of the
29 tissue types.
Tukiainen also came across something surprising in the GTEx
database. Normally, X inactivation happens randomly to one X
chromosome in each cell, so most XX individuals are a mosaic of
cells with one or the other X shut down. One of the project’s tissueThe concept is out there that genes that
escape X inactivation are infl uencing
female-specifi c physiology.
—Sundeep Kalantry, University of Michigan Medical School