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Cantone Reversal of X chromosome inactivation

3 days after fusion) but not in all of them (50% at day 3). somatic cells with low frequency and is enhanced upon
XACT instead re-associates with either one or two X reprogramming. Interestingly, recent studies of three-
chromosomes later during reprogramming in a minority dimensional X chromosome organization in mouse
of both mono-allelic and bi-allelic cells (< 1% at day 6). suggested that genes variably expressed from the Xi
These results suggest that human Xi reactivation is in a cell-specific context co-localize within topologically
induced by cell fusion-mediated reprogramming ahead, associated domains and showed reduced expression
or immediately after, cell division and that it requires upon disruption of the domain architecture [131] . As loci
XIST delocalization, but not XACT re-association along that escape X inactivation vary between different cell
the Xi. Notably, delocalization of XIST upon cell fusion- types and also between females [10] , future population
mediated reprogramming results in a diffuse nuclear analysis of somatic cells from different tissues will help
signal that resembles the one observed in human us to understand the molecular basis for this higher
blastocysts and might represent remaining binding reactivation susceptibility.
at foci from where XIST initially spreads [127,128] . In
addition, a minority of heterokaryon and hybrid nuclei Another important finding of our Xi reactivation
(7% at day 3 vs. 1% at day 0; unpublished data) have study is the possibility of using cell fusion-mediated
two diffused XIST clouds suggesting that cell fusion- reprogramming to identify distinct subsets of Xi
mediated reprogramming might be able to recapitulate genes based on their susceptibility to reactivation
some features of the in vivo epiblast cells. Consistently upon further epigenetic perturbations. Indeed, DNA
with this hypothesis, genome-wide expression analysis demethylation of human fibroblasts allowed us to
upon cell fusion-mediated reprogramming showed identify a second subset of genes that was reactivated
the reactivation of pluripotency genes associated with only after reprogramming. This suggests that the
both primed and naïve human pluripotent cells [129] . In extensive Xi chromatin remodeling that takes place
the future, single cell studies might indeed be used to during reprogramming might unmask limiting factors in
segregate different human pluripotent states and help the reactivation of Xi gene subsets.
us refine culture conditions for naïve pluripotency.
FINAL REMARKS
To investigate the extent of Xi gene reactivation
along the entire X chromosome, we derived human Pluripotent reprogramming of somatic cells has been
fibroblast clones with reciprocal Xi chromosomes widely used to induce the reversal of X chromosome
2
1
1
2
(e.g. Xa Xi and Xi Xa ) and performed allele-specific inactivation and investigate its molecular mechanisms.
RNA-seq analysis of these clones before and after In the last decade, mouse somatic cell reprogramming
reprogramming [129] . This analysis showed that cell allowed major steps forward in unraveling the molecular
fusion-mediated reprogramming induces partial human connections between pluripotency and X chromosome
Xi reactivation with 10% of the sampled genes being inactivation/reactivation [51,52,71,98] . Reprogramming,
consistently reactivated at different times after fusion indeed, represents a promising system to investigate
and in different clones. Notably, clones with opposite Xi the functional role of newly discovered XCI factors [23-27] .
haplotype reactivate the same set of genes suggesting
that neither parent of origin nor mutations in regulatory In light of recent findings in human embryos, the
sequences influence reactivation. We instead observed interpretation of mouse XCI studies need to be
a preferential reactivation of Xi genes localized within carefully considered when translated to human. Recent
XCI “escape” domains at the telomeric end of the short analyses of human pre-implantation development show
Xp arm suggesting a higher susceptibility of these remarkable differences between the events and the
loci to reactivation. Similar results were obtained in a molecular players that characterize X chromosome
large iPSC population study that identified Xi domains inactivation in human and mouse, suggesting unforeseen
reactivating with higher frequency [130] . Conversely to specie-specific mechanisms [60,61,103] [Table 1]. In addition,
iPSC erosion, however, Xi genes that were reactivated the epigenetic instability and heterogeneity of human
upon cell fusion-mediated reprogramming could not pluripotent cells upon time in culture limit the possibility
be predicted by H3K27me3 domains suggesting of modelling human XCI and its reversal in vitro.
that the mechanism leading to reactivation might be Recently, comparison of transcriptome profiles of single
different in these two systems [107] . Cell fusion-mediated epiblast cells with human pluripotent cells allowed the
Xi reactivation could instead be predicted by variable identification of culture conditions that better preserve
Xi expression among single cell-derived fibroblast naïve pluripotency ex vivo [116,122,123] . XCI state, however,
clones ahead of reprogramming. This probably cannot be fully recapitulated in these cultures [62,103] thus
reflects an intrinsic predisposition of certain loci to highlighting the need for further improvements. We
transcriptional activation that can already occur in the propose that cell fusion-mediated reprogramming might

Journal of Translational Genetics and Genomics ¦ Volume 1 ¦ November 16, 2017 9