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Cantone Reversal of X chromosome inactivation

or structural abnormalities is often found as inactive future to engineer selective Xi gene reactivation as a
in the majority of somatic cells due to selection [3,4] . novel clinical approach for several X-linked diseases.
Many X-linked diseases are indeed asymptomatic in
heterozygous females and manifest only in their male Our current understanding of XCI and its molecular
progeny . However, some X-linked mutations are lethal mechanisms derives from studies in the mouse. During
[5]
in males and lead to severe disease in heterozygous mouse embryonic development, Xi gene silencing is
females . For example, in the case of gene mutations triggered by Xist, a long non-coding RNA that is up-
[6]
with a dominant negative effect, expression of a wild- regulated from the future Xi and spreads along the
type allele from 50% of cells is not able to rescue chromosome domain [22] . Although the repertoire of Xist
the function in heterozygosity. This is exemplified interacting proteins has been recently identified, the
by UBQLN2 mutations that cause abnormal protein precise molecular mechanisms by which Xist initiates
aggregation and consequent neurodegeneration in silencing are still unknown [23-27] . It is currently believed
X-linked amyotrophic lateral sclerosis . Importantly, that Xist might act as a scaffold to recruit further
[7]
there are other diseases in which the compensatory factors on the Xi, including Polycomb repressive
effect of XCI in heterozygous females is not effective complex 1 (PRC1), histone deacetylases, histone
due to tissue-specific sensitivity to dosage unbalance variants and the DNA methylation machinery [24,25,28-30] .
and/or to skewed XCI patterns that favor the expression Upon Xist coating the inactive X chromosome indeed
of mutated genes. For example, expression of the wild- becomes progressively devoid of RNA polymerase II
type allele only from half of the cells may lead to loss of and chromatin marks associated with transcriptionally
function only in certain tissues but not in others, as for active regions [31] ; whereas it is enriched of repressive
mutations in the X-linked MECP2 gene that specifically marks such as H3K27me3 [32] , H3K9me2/me3 [33] ,
cause a severe brain deficit even if the gene is H2AK119ub [34,35] , macroH2A1 [36] and promoter
constitutively expressed [8,9] . In addition, in human some DNA methylation [37] . The multitude of epigenetic
genes escape inactivation and are variably expressed modifications that are deposited along the Xi are
from the Xi in different tissues of the same individual and believed to maintain silencing in a redundant manner
even in-between different females [10] . This variability as removing single factors does not lead to global
in Xi expression has been suggested to influence the gene reactivation. Supporting this hypothesis, it has
penetrance and expressivity of X-linked diseases as been shown that Xi gene silencing is maintained in
hypothesized for oro-facial-digital syndrome type 1 . the absence of macroH2A1 histone variant [38] and
[11]
Finally, it has been shown that XCI skewing can also upon functional loss of Polycomb repressive complex
favor cells in which the wild-type allele is on the inactive PRC2 and some PRC1 components, which catalyze
X chromosome thus leading to the outbreak of disease the deposition of the repressive chromatin marks
in heterozygous females [12-16] . H3K27me3 and H2AK119ub respectively [39,40] . A
recent study of several Xist interacting proteins has
An important implication of the epigenetic regulation also shown that knock-down of any single interactor
of Xi gene expression is the preservation of the could not reactivate an Xi-integrated GFP reporter,
genetic material and, indeed, the presence of a whereas combining targeting of each interactor with
silent set of alleles for over a thousand genes on the inhibition of DNA methylation and topoisomerases
X chromosome. Reactivation of wild-type alleles in leads to reactivation of 75-100 Xi genes out of around
heterozygous females could potentially be harnessed 200 analyzed [25] . On the other hand, depletion of Xist in
and might represent a therapeutic approach for many somatic cells leads to a stochastic reactivation of single
X-linked diseases. This is probably best exemplified Xi genes [41] that can be potentiated when combined
by Rett syndrome, for which a phenotypic reversal of with histone deacetylase (HDAC) inhibitors and DNA
advanced neurological symptoms has been shown demethylation [42] . Importantly, Xist depletion in mouse
in both young and adult mice upon expression fibroblasts has been shown to alter the Xi chromosome
of a wild-type MeCP2 [17] . Importantly, rescue of conformation making it more similar to the one of the
disease symptoms has also been observed in active X chromosome (Xa) [25] . In addition, silencing of
human and mouse upon gene therapy of X-linked factors that affect Xist expression and/or localization
adrenoleukodystrophy [18] and Hunter syndrome [19,20] , has been shown to partially reactivate the Xi [43,44] .
two recessive X-linked diseases that also affect Altogether these findings support the hypothesis that
heterozygous females [16,21] . This suggests that the multiple epigenetic layers are in place to prevent Xi
usefulness of Xi reactivation in clinical approaches reactivation and suggest a central role for Xist both in
might be extended beyond dominant X-linked diseases. the initiation and maintenance of XCI.
Understanding the molecular mechanisms of Xi
inactivation and reactivation will be instrumental in the Cell fate reprogramming studies offer the opportunity

2 Journal of Translational Genetics and Genomics ¦ Volume 1 ¦ November 16, 2017