03.2020 | THE SCIENTIST 27when analyzing human datasets: binding sites for the YY1 transcrip-
tion factor were common near escapees.^12
The escape and boundary signals appear to be similar in mouse
and human DNA, Brown’s group found in experiments mirroring
Carrel’s. The team isolated a snippet of human X DNA containing
three genes: the escapee RPS4X, the normally silenced ERCC6L, and
the variably expressed CITED1. They placed this segment in a mouse
X, at a silenced region, and the human genes’ expression patterns
held.^13 The results reinforced Brown’s belief that there must be pro-
escape signals in the escapee genes—signals that mouse cells can read.
But it’s still difficult to predict from sequence alone if a gene
will escape or not. For every potential marker, there are genes that
buck the trend, remaining silent despite suspected escape signals.
“We haven’t come up with a rule,” says Brown. “It’s also possible that
there’s some unique combination of elements, so not just a single
element, but multiple ones.”
To find the rules, she’s now dissecting RPS4X, taking out suspect
elements one by one to learn how they influence escape. She’s also
attempting to assemble a wholly artificial escapee, starting with a
promoter and adding candidate escape elements such as YY1 bind-
ing sites. Then, Brown says, “we can actually test whether or not
they’re going to favor escape.”
How X genes mediate silencing
Some Barr body genes control X inactivation itself.XIST, first
described in 1991 by Brown,^14 is one; the long noncoding RNA
transcribed from the gene is still considered the primary factor in
X inactivation. But it might not be the only involved gene located
on the inactive X chromosome; Kalantry recently discovered evi-
dence for another.
To study the function of Xist (the murine version of the gene) in
mouse embryos, Kalantry took advantage of the gene’s own regula-
tory system. If RNA polymerase reads Xist in the antisense direc-
tion, it produces a transcript called Tsix (Xist backwards), some-
how blocking production of Xist RNA. Kalantry used a strain with a
mutation that cut short Tsix transcription, giving Xist full rein. This
triggered the inactivation of both X chromosomes in cells of female
embryos and stem cell lines. Not surprisingly, this killed those cells.
But the Tsix mutation did not effectively shut down the sole X
chromosome in XY cells. Kalantry reasoned that some factor on the
Y chromosome might block X inactivation. That hypothesis proved
false when he looked at embryonic stem cells with only one X and
no Y, what’s called an XO genotype, and saw the same pattern: the
X chromosome remained active.^15
Kalantry then hypothesized that something in the XX cells
permitted inactivation. These cells already contained one properly
inactivated X, plus the active one Kalantry was trying to repress.
Could some escapee from the inactive X be enabling inactivation
of one or both chromosomes?
In a preprint posted on bioRxiv in 2017, Kalantry and col-
leagues described a candidate for this silencer: Kdm5c, the escapee
from Carrel’s studies.^16 It encodes a transcription factor that can
turn genes on or off by removing methyl groups from the local his-
tones. That fits what Kalantry believes the KDM5C protein does:
turn Xist on, but other X genes off.
When the researchers knocked out Kdm5c in mouse embryos,
females showed little to no X inactivation, and subsequently died.
With female cells that had only one copy of Kdm5c, inactivation took
place, but some normally silenced genes on the inactive X began to
speak up. Engineering XY embryonic stem cells to overexpress the
gene resulted in the inactivation of their only X, and they subsequently
died. Thus, in both male and female embryos, full X inactivation
appears to require more than one gene’s worth of KDM5C.
To follow KDM5C in the cell during X inactivation, the team
watched it using immunofluorescence. As Xist RNA coated the X
chromosome, KDM5C started to show up as well. Once the Barr
body was formed, most KDM5C disappeared, but further experiments
showed that a bit of the protein persisted around promoters and other
regulatory elements.Kalantry posits that KDM5C could regulate inactivation as fol-
lows: Early in embryogenesis, before inactivation, XX embryos make
a high dose of KDM5C, expressed from both chromosomes. At this
concentration, the transcription factor activates Xist, while helping
to silence other genes on one X chromosome. After the Barr body is
formed, Kdm5c continues to escape, maintaining that silence. Now,
he’s looking to see if the actions of KDM5C are similar in other species.Escapee function for good and ill
Besides seeking to better understand the process of X inactiva-
tion, researchers studying escape want to know what all these
unsilenced genes are doing in the cell, and whether they matter
for human health. Escapee expression, which would create pro-
tein level differences between XX and XY individuals, may explain
why some conditions are more prevalent in one sex or the other.
Multiple sclerosis (MS), a disease in which the immune system
attacks the insulating myelin around nerve fibers, is about three
times more common in women than men. Sex hormones seem to
explain some of the increased risk in women, but not all of it. Rhonda
Voskuhl, director of the University of California, Los Angeles (UCLA),
MS program, wondered if the XX or XY genotype might contribute.
She partnered with UCLA geneticist Arthur Arnold to work
with a set of mouse lines he’d created to disassociate the effects of
sex chromosomes from those of gonads and sex hormones. The
lines include XX animals with male gonads and hormones, and
XY mice with female gonads and hormones. Together with male
and female mice with the usual combinations of sex chromosomes,
hormones, and gonads, these form the four core genotype models.^17
Using them, researchers can control for the effects of sex hormones
to elucidate the role of sex chromosomes alone.
Voskuhl immunized the four core mice against a component of
myelin so their immune systems would attack it, a standard methodSome Barr body genes, including XIST
itself, actively control the silencing of
other stretches of the X.