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Cantone Reversal of X chromosome inactivation

mESCs were used as donors for nuclear transfer. declining from late S through mitosis [94] . Nonetheless,
This most likely results from the failure in resetting macroH2A is retained on the Xi during mitosis whereas
imprinting marks of donor nuclei [88] , which are instead human XIST and other Xi-associated histone marks
erased during normal development in primordial germ (e.g. H2A ubiquitination) are lost [95] . As cell cycle has
cells and re-established in the gametes in a parent- been shown to influence the efficiency of SCNT-
specific manner [89] . Further studies confirmed that reprogramming (discussed above) and mouse Xist is
cloned embryos fail to establish imprinted XCI in instead associated with the Xi throughout mitosis, it will
preimplantation stages and extraembryonic tissues, be interesting to determine whether the developmental
and revealed heterogeneity in random XCI within state of the donor nucleus and/or its cell cycle
cloned embryos in which some cells undergo XCI phase also influence Xi reactivation and investigate
while others do not inactivate any X chromosome [90] . mechanistic differences between mouse and human.
Although progressive loss of Xist coating has been
observed within 30 min after SCNT, the precocious iPSC-mediated Xi reactivation
appearance of H3K27me3 and H3K9me2 on the Global epigenetic resetting has been observed upon
original Xi suggests an incomplete reprogramming induction of pluripotency by transduction of four
of the somatic nucleus [91] . The extent of Xi gene pluripotency factors (i.e. Oct4, Sox2, Klf4 and c-Myc)
reactivation remains however unknown. Altogether into mouse fibroblasts [96,97] . Similarly to mESCs,
these studies suggest that nuclear transfer cannot miPSCs have two active X chromosomes and undergo
fully erase Xi epigenetic marks during pre-implantation de novo random XCI upon differentiation [97] . Recent
development and further reprogramming events are studies have indeed used iPSC reprogramming to
required for re-establishing the normal developmental dissect the molecular mechanisms of Xi reactivation in
program. Interestingly, ectopic Xist accumulation has mouse [52,98] .
been observed upon SCNT in both male and female
embryos and loss or depletion of Xist have been A tight association between the reversal of XCI and the
associated with increased efficiency of reproductive sequential activation of pluripotency factors has been
cloning [92,93] . In order to get some mechanistic detected by investigating the kinetics of Xi epigenetic
insights, it would be important to determine to which changes during mouse iPSC reprogramming [52] .
extent genes along the Xi are reactivated in single Specifically, it has been shown that loss of Xist from the
blastomeres and their correlation with the observed Xi Xi follows Nanog expression, consistently with Nanog
epigenetic changes. role in repressing Xist expression. Xi gene reactivation
was instead observed in a subset of Nanog positive
Xi reactivation has also been studied by injecting cells that reactivate additional factors (i.e. DPPA4
somatic nuclei into the germinal vesicle of Xenopus and PECAM1) at later reprogramming stages. This
oocytes [51] . This study showed that the developmental suggests that the hierarchical activation of pluripotency
state of the donor influences the reactivation of an factors is required for complete reversal of XCI [52,99] .
X-linked transgene located on the Xi. Specifically, Consistently with this hypothesis, depletion of Nanog
reactivation was observed when mouse post- impaired Xi reactivation, whereas its overexpression
implantation epiblast stem cells (EpiSCs) were injected during late reprogramming stages promoted the
into frog oocytes but not upon transfer of embryonic formation of iPSC colonies expressing DPPA4 and bi-
fibroblasts or extraembryonic cell nuclei. Comparative allelically transcribing Tsix from both X chromosomes.
Xi chromatin analysis showed loss of Xi-associated These data suggest that Nanog expression is required
Xist both in EpiSCs and reactivation-resistant cells, but not sufficient for efficient reversal of XCI. A further
whereas H3K27me3 and DNA methylation were link between pluripotency and Xi reactivation has
invariably maintained. Accumulation of the histone been provided by Prdm14, a germline factor that has
variant macroH2A was instead observed on the been implicated in the epigenetic reprogramming
Xi of reactivation-resistant cells but not in EpiSCs. of PGCs and whose expression correlates with Xi
MacroH2A depletion upon transfer of fibroblast reactivation [100] . It has been shown that depletion
nuclei lead to partial Xi reactivation that could be of Prdm14 during iPSC reprogramming decreases
enhanced when it was combined with HDAC inhibitors Xi reactivation and hampers both the derivation
or activation of Oct4 and Sox2. This suggests that and maintenance of iPSC colonies [98] . Prdm14
macroH2A contributes to the stability of Xi but overexpression in mouse EpiSCs instead induced
other factors are required for full Xi reactivation. efficient conversion to ESCs and Xi reactivation [101] .
Notably, it has been shown that in human somatic Mechanistically, Prdm14 has been shown to repress
cells macroH2A association with the Xi is cell-cycle Xist in a dual manner. First, it represses Rnf12, a E3-
dependent being most prominent in early S phase and ubiquitin ligase that targets Rex1 for proteosome

Journal of Translational Genetics and Genomics ¦ Volume 1 ¦ November 16, 2017 5