| Summary: | X-chromosome inactivation is the transcriptional silencing of one of the two X
chromosomes in the mammalian female and is thought to be a means of dosage compensation.
The inactive X chromosome is condensed, late replicating, hypoacetylated and hypermethylated
at CpG islands. The silenced chromosome also expresses Xist - a fascinating large, untranslated
RNA involved in the inactivation process. Although the mechanism of Xist function remains
unknown, it has been established that the RNA is necessary for X-chromosome inactivation in
two eutherian mammals - humans and mice.
Xist fragments have also been identified for several other eutherians, but the only
complete sequences are those of the human, murine and vole genes. Interestingly, Xist has not
yet been identified in marsupials, although these mammals undergo X-silencing as well.
Imprinted inactivation and the lack of CpG methylation are two additional aspects of X -
inactivation in which the marsupials differ from the eutherians. X-chromosome inactivation
studies in the eutherian mammals have mostly been limited to the human and murine model
organisms and little is known about the process in other members of this mammalian subclass.
This study examined the process of X-inactivation and identified the Xist gene in a
virtually unstudied mammalian organism - the Coast mole, Scapanus orarius. The mole
belongs to the order Insectivora, an ancient and possibly basal mammalian group, which was
traditionally thought to be closest to the eutherian common ancestor. Prior to this study, X-chromosome inactivation in the insectivores had not been investigated in detail and the Xist
gene had not been identified. By using methylation-sensitive restriction digests of several X-linked genes (ARA, FMR1 and ZFX) followed by PCR with primers flanking the restriction
sites, this study confirmed the presence of a methylated X chromosome, indicating that these
animals undergo X-inactivation. Additionally, the ARA and FMR1 genes were found to be
subject to silencing, while ZFX was shown to escape X-inactivation in the mole. The results of
the methylation assay not only show that X-inactivation exists in the Coast mole, but that
silencing through methylation is conserved within the Eutheria.
Low stringency PCR with primers designed from sequence conserved among the
available Xist genes was successful in amplifying a mole fragment. Sequence analysis and
female-specific expression confirmed that this was the Coast mole Xist homologue. Additional
Scapanus Xist fragments were obtained through long range PCR and inverse PCR resulting in a
total of 2.5 kilobases of sequence, which show significant similarity to Xist fragments from
other mammals. The presence of Xist in the mole, along with its exclusively female expression
pattern and sequence similarity to the gene in other species, indicate that Xist is involved in the
X-inactivation process in this insectivore.
In conclusion, the study of X-chromosome inactivation in the Coast mole shows that the
unique features of eutherian X-linked silencing have been conserved in the Order Insectivora.
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