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Cantone Reversal of X chromosome inactivation

X chromosomes. by directly repressing Xist and activating Tsix.
Consistently with this hypothesis, re-expression of Oct4
Cell fusion-mediated Xi reactivation from the somatic genome has been shown to precede
The earliest evidence of an association between Xi reactivation and repression of Xist expression upon
reversal of XCI and pluripotent reprogramming of somatic cell fusion-mediated reprogramming [50] . The delayed
[68]
cells is dated back to 1983 when Takagi et al. showed repression of Xist has been associated with the kinetics
that fusions between female mouse thymocytes and of DNA methylation at its 5’ regulatory sequences
pluripotent mouse embryonic carcinoma cells (ECCs) and is enhanced by the activation of Dnmt3a and Tsix
shifted the replication timing of the thymocyte Xi from via HDAC inhibitors. This suggests that chromatin
late to early S phase, as observed for transcriptionally remodeling is required together with pluripotency factors
active X chromosomes. Early replication timing was in order to reverse XCI, although delocalization of Xist
associated with reactivation of the X-linked Pgk1 gene rather that loss of its expression has been associated
from the Xi and was observed upon fusions of several with Xi gene reactivation [79] .
differentiated cells from thymus, spleen or bone
marrow with ECCs, but not when two differentiated Somatic cell nuclear transfer-mediated Xi
cell types were fused together. Importantly, hybrids reactivation
obtained between mouse somatic cells and ECCs The somatic cell genome can restart the entire
acquired the pluripotent differentiation potential developmental program upon nuclear transfer into
of the parental carcinoma cells suggesting that eggs or oocytes. Conversely to other reprogramming
reprogramming towards pluripotency might trigger Xi techniques, somatic cell nuclear transfer (SCNT)
reactivation. Later studies confirmed that embryonic reprograms differentiated cells to a totipotent state from
pluripotent cells of different origin (including ECC, which both embryonic and extra-embryonic lineages
ESC and embryonic germ cells, EGC) are able to develop into cloned animals [80,81] . In this model system,
reprogram the somatic cell genome and reverse XCI reprogramming is triggered by maternally-inherited
upon fusion. Specifically, it has been shown that the factors that are present in the cytoplasm of metaphase
somatic Xi acquires epigenetic features of the active II oocytes. Interestingly, it has been shown that the
X chromosome (Xa), including early replication timing, zygote and blastomeres of early embryonic stages are
loss of Xist coating and Xi-associated histone marks not capable of reprogramming somatic nuclei unless
(e.g. H3K27me3 and H2Aub1), and re-expresses an they are in mitosis [82,83] . The enhanced reprogramming
Xi-linked GFP transgene [69-71] . Importantly, somatic- capacity of mitotic cells has been ascribed to the
ESC hybrids are able to re-initiate random XCI upon release of chromatin-bound factors during mitosis and
differentiation suggesting a complete erasure of depends upon cell cycle synchronization between
epigenetic memory along the Xi [70,72] . However, the the somatic donor and the embryonic recipient [84,85] .
fusion of ESC with somatic cells does not lead to This evidence suggested a model in which the mitotic
erasure of genomic imprinting [69,73] . This extended environment of the recipient cell induces premature
reprogramming potential has instead been observed in chromatin condensation and consequent release
fusions with EGCs that can induce both Xi reactivation of chromatin-bound factors in somatic nuclei, thus
and loss of DNA-methylation imprinting marks in the facilitating genomic access of transcriptional regulators
somatic nuclei [73,74] . that re-establish a totipotent transcriptional program [86] .

Cell fusion studies have also demonstrated that the The success of nuclear reprogramming has been
reprogramming capacity of embryonic pluripotent cells associated with erasure of epigenetic memory from
is ascribed to the transfer of pluripotency-associated the somatic nucleus [87] and the reactivation of the
transcriptional factors into the somatic nucleus [71,75] . somatic Xi has been used as a model to investigate
Specifically, it has been shown that mouse ESCs epigenetic reprogramming. Initial experiments by
(mESCs) overexpressing Nanog have an enhanced Eggan et al. [49] showed that the reactivation of a GFP
reprogramming capacity whereas ESCs lacking Oct4, transgene on the somatic Xi occurs by the morula/
but not Sox2, fail to induce pluripotent reprogramming blastocyst stage and is followed by random XCI in
in the somatic fusion partner. This is consistent with the embryonic lineages. In the extraembryonic cells,
findings in mESCs where Oct4, Nanog, Sox2 and instead, the somatic Xi is re-inactivated suggesting
Rex1 repress Xist expression, while Klf4, c-Myc an incomplete erasure of epigenetic memory in this
and Rex1 up-regulate Tsix [45-48] , a long noncoding lineage, in which imprinted inactivation of the paternal
RNA that antagonizes Xist and protects the Xa from X chromosome occurs during normal development.
inactivation [76-78] . These results suggest that pluripotency Consistently with this hypothesis, random XCI was
factors within the mESCs might induce Xi reactivation observed in the extraembryonic lineages when

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