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Page 4 of 15 Przanowski et al. J Transl Genet Genom 2018;2:2 I http://dx.doi.org/10.20517/jtgg.2017.03

pointed to more direct role of PRC1 in XCI, thereby pointing to a non-canonical recruitment of PRC to
Xi . Some of these differences can be attributed to cell lineages used in the different studies or the technical
[28]
approaches; however more in depth investigation is needed to better understand the PRC-mediated silencing
of Xi. The discrepancies in the conclusion of various studies regarding the role of PRC in XCI have been
[29]
discussed in details elsewhere and are not discussed further in this review. DNA methylation has also
been found to play an important role in the stabilization of the Xi state . In summary, the plethora of
[30]
epigenetic modifications accumulating on Xi define its higher-order organization, which is essential for
stabilizing the three dimensional structure of Xi .
[31]

Previously, XCI has been considered unidirectional during later stages of development, but more recently,
the idea of reactivating Xi has emerged. Evidence suggests that the epigenetic state of Xi can be reversed by
genetic or pharmacological manipulations [32-34] . Although these results were paradigm shifting, they were
not unexpected due to the inherent ability of Xi to shuttle between the active and inactive states during
embryogenesis. During embryo formation, XCI occurs in two distinct stages: first, imprinted inactivation
of paternal X (Xp), and second, random inactivation of either paternal (Xp) or maternal X (Xm) upon
differentiation [35,36] . Xp is inactivated shortly after fertilization due to maternal imprint, but it is subsequently
reactivated at the blastocyst stage of embryo development. This process is followed by random XCI in the
cells of the inner cell mass (ICM) of the embryo, and maintained throughout mitosis . Extensive analysis
[37]
using in vitro differentiation of cells isolated from ICM has enabled a comprehensive understanding of the
early events in the random XCI [Figure 1]. Further, the analysis of XCI status in epigenetically stable basal
[38]
breast cancer and ovarian cancer cell lines have revealed defects in Xi, evident by cells carrying two Xa .
Although a preferential loss of Xi and gain of an additional copy of Xa could explain the Xa polyploidy,
the reactivation of Xi could not be ruled out. In fact, by identifying single nucleotide polymorphism (SNP)
differences in the X-linked genes, it was demonstrated that Xi was indeed reactivated in a subset of breast
[39]
cancer cells . Together, these findings point to the tantalizing possibility of synthetically manipulating the
state of Xi, which could have far reaching therapeutic implications for monogenic X-linked diseases, such as
Rett syndrome, CDKL5 syndrome, and Fragile X syndrome. The reversal of XCI could compensate for the
gene deficiencies by expressing the endogenous wild-type copy from Xi. Several studies have demonstrated
that upon the interference with the function of regulatory factors of XCI, the Xi can be reactivated in
mouse embryonic fibroblasts, fibroblast cell lines and cortical neurons [32-34] . However, before embarking on a
translational approach based on the idea of X chromosome reactivation (XCR), a detailed understanding of
the XCR mechanism and regulatory factors is warranted.

Here, we review recent advancements in the XCI field that has provided crucial insights into the mechanism
of XCI and the regulators of the epigenetically inert landscape of Xi. We also discuss different methodologies
that combine biochemical, genomics, genetic and proteomics strategies to investigate the mechanism of XCI.
Using these new multidisciplinary technological strategies, we are better equipped with tools that can guide
the reversal of the established inactive state of the X chromosome. Identification of XCI factors (XCIFs) and
their individual roles in the silencing process are essential for comprehension of the mechanism as a whole
and could potentially aid development of novel approaches to treat X-linked diseases.

RECENT TECHNOLOGICAL ADVANCES PROVIDING NEW MECHANISTIC INSIGHTS INTO THE
XCI
A high level of epigenetic stringency of XCI accentuates the fascination with this paradigmatic phenomenon,
but at the same time its complexity has contributed to the stalled progress. The silencing of mammalian
X chromosome requires a well-regulated cascade of chromatin changes that are orchestrated by a battery
of XCI regulators, which include both cis and trans-acting protein-coding as well as non-coding RNAs.
Several of these crucial regulators remain unknown, mainly due to technical difficulties in discriminating