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Cantone Reversal of X chromosome inactivation

chromosome in XaXi class II hESCs and in the human for hiPSC that were initially supposed to reactivate the
epiblast ahead of XCI, it has been suggested to Xi upon culture on LIF producing feeders. The latter
antagonize XIST by competing for Xi binding or limiting have instead been shown to undergo extensive erosion
chromosome accessibility [103,109] . Consistently with this of XCI by multi-gene RNA-FISH [57] .
hypothesis, overexpression of a XACT transgene in
mESCs has been shown to prevent inactivation of the Recently, two culture conditions [116,122] have been
X chromosome from which it is expressed, whereas its shown to reprogram hESCs and hiPSCs to a state
downregulation restores random XCI [103] . similar to human blastocysts [123] . Detailed analysis
of XCI state in these “naive” human pluripotent
The variable XCI state and its progressive epigenetic cells showed that they retain some features of the
alterations in hESCs have been suggested to result epiblast, including dual XIST and XACT coating, bi-
from inappropriate culture conditions that are unable allelic expression of X-linked genes and dampening of
to stabilize the XCI state during the derivation from X-linked gene expression on both X chromosomes [62,103]
the in vivo epiblast and further in vitro passaging [111] . [Figure 2B]. However, the same Xi was re-inactivated
Derivation in low oxygen levels allowed to obtain hESCs upon differentiation and only a minority of cells within
with two active X chromosomes and was suggested the culture (< 10%) showed XIST-coating on both X
to preserve this state [112] . However, a separate study chromosomes. Collectively, these results suggest that
showed that hypoxia rather stabilize hESCs that have an improvement of culture conditions is still required
already undergone XCI [113] . Other studies reported the for stabilizing human naïve pluripotency and careful X
derivation of hESCs that preserved the ground state chromosome-wide analysis of Xi-specific expression
of the in vivo epiblast but they did not fully characterize need to be performed in the future to unequivocally
the status of the X chromosomes [114-116] . In light of define XCI state in pluripotent cells.
recent findings showing that the two X chromosomes in
human female embryos are characterized by dual XIST Xi reactivation by interspecies cell fusion-
coating and dampening of X chromosome expression, a mediated reprogramming
recent work highlights the importance of characterizing Cell fusion between somatic and ESCs from different
chromosome-wide X-linked gene expression and XIST species has been used to investigate human pluripotent
nuclear localization before and after differentiation [57] . reprogramming [75,124] . This system allows the analysis of
Multi-gene RNA-FISH and allele-specific X-linked gene early reprogramming events because specie-specific
expression revealed that hESC derived and propagated features in nuclear organization (e.g. the presence of
in standard FGF2-containing medium maintain their XCI chromo centers in mouse nuclei) and DNA sequence
state upon differentiation. Notably, it has been shown differences can be used to track each fusion partner
-
that XIST XaXa hESCs (previously defined as class by imaging and molecular techniques. Importantly, cell
I) cannot re-express XIST neither undergo XCI upon nuclei remain separated within a shared cytoplasm and
differentiation similarly to cells in the XaXe eroded state. this transient heterokaryon state persists until the first
These aberrant cells arise from blastocyst outgrowth mitosis, when nuclei fuse and generate hybrids [125] . We
as early as 48 h after plating onto feeders and can be have, indeed, recently used cell fusion between human
stably maintained in this state upon establishment and female fibroblasts and mouse ESCs to reprogram
propagation of hESCs. Although it cannot be formally the somatic nucleus and investigate human Xi
excluded that these cells represent an intermediate reactivation [126] [Figure 2C]. We showed that expression
state in human XCI, they are currently believed to result of pluripotency genes from the human nucleus occurs
from epigenetic adaptation to in vitro culture. as early as two days after fusion at a time when the
majority of cells are heterokaryons. This observation
Similar epigenetic instability has been shown to allowed us to discriminate pre- and post-mitotic
occur in human iPSCs and it is probably the cause of reprogramming events in heterokaryons and hybrids,
controversial results in different labs [58,59,117-121] . Some respectively. Single cell analyses demonstrated that
groups, in fact, reported that XCI is maintained upon XIST delocalization and loss of H3K27me3-enrichment
human iPSC reprogramming [58,59,117,119] while others from the human Xi occur in heterokaryons and hybrids
claimed Xi reactivation [55,118,120,121] . Most of these studies, 2-3 days after fusion, and precede bi-allelic expression
however, analyzed indirect markers of Xi reactivation, of ATRX and HUWE1, two X-linked genes that are not
such as XIST and H3K27me3 nuclear localization and subject to XCI erosion in human ESCs [57,107] . RNA-FISH
expression of X-linked genes compared to autosomes, analysis of nascent ATRX and HUWE1 transcripts
or directly assessed allele-specific expression of only together with XIST or XACT RNAs showed that bi-
few Xi genes. These analyses could easily confuse Xi allelic expression of X-linked genes only occurs in cells
reactivation with erosion, as it has been recently shown that have lost a localized XIST signal (i.e. about 30% at

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