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

balances gene dosage by ensuring that only one X chromosome is transcriptionally active per diploid cell.
However, the molecular details of the well-coordinated silencing of the X chromosome are not completely
understood.

The mammalian XCI is a multistep process and requires multiple regulatory factors for its successful execution.
However, gene dosage compensation is not unique to mammals, but also occurs in invertebrate species, such
as Drosophila melanogaster and Caenorhabditis elegans. While D. melanogaster doubles transcription from
the X chromosome in XY males, the X-linked gene expression is halved in XX C. elegans females to equalize
[2,3]
gene expression amongst genders . Even though the mechanistic strategies are substantially different
among species, considerable parallels are observed in the mechanisms by which dosage compensation is
achieved. Based on the investigation in different models, XCI has three well-demarcated stages: initiation,
establishment and maintenance . Lack of molecular details has been a major roadblock in understanding
[4]
how this complex process of XCI is regulated. The key players required for maintaining stringent control of
the transcriptional state of only one X chromosome in female cells remain mostly unidentified. Importantly,
defining the mechanism of XCI will also shed light on some of the unaddressed questions in the field.
For instance, one of the major challenges is how the compensation machinery discriminates between two
essentially identical X chromosomes. What also remains unanswered is the mechanism that precisely controls
the counting of the X chromosomes and the random choice of only one X chromosome for inactivation. How
does proper initiation and spreading of Xist along the X chromosome occur? The identification of regulatory
factors uniquely interacting with the silent X chromosome at various steps may also provide much needed
insight into the XCI mechanism.

While the mechanism of XCI is not completely defined, Xist is one of the very well-characterized regulatory
factors central to XCI. Xist is a 17kb long non-coding RNA (lncRNA) whose expression is very tightly regulated
in a cell-stage specific manner. The stable monoallelic Xist expression marks the initiation of the silencing
of Xi and coats the chromosome in cis . Mouse embryonic stem cells (ESCs) have served as a model system
[5]
[6,7]
to dissect the early steps of XCI, because mouse ESCs carry two active X chromosomes (Xa) . But at the
onset of differentiation, the Xist transcription is upregulated on Xi along with several other stochastic
[8]
fluctuations in regulatory factors, such as pluripotency factors and epigenetic regulators . While Xist is
essential for the initiation of XCI, an antisense non-coding RNA, Tsix plays a crucial role in preventing Xist
expression from the Xa . Although the mechanistic details of Tsix-mediated Xist down-regulation are not
[9]
completely understood, a recent study showed that the Hedgehog paracrine system induces Tsix expression
[10]
in pluripotent stem cells . Another study suggested that Xist down-regulation could result from either
binding of transcriptional factors within Tsix, or from counter-current movement of a Polymerase complex
that would inhibit production of a sense Xist transcript [9,11,12] . Alternatively, Tsix itself could be a functional
repressor by sequestering Xist and targeting Xist for degradation by RNA interference (RNAi) dependent
mechanisms . Once XCI is established, Tsix is only transcribed at low level from the Xa. Additional
[12]
repressors of Xist expression include the pluripotency factors, such as SOX2, OCT4, NANOG, and PRDM14,
which prevent XCI in undifferentiated ESCs . Down-regulation of the pluripotent factors precedes the
[13]
initiation of XCI. Several positive X-encoded activators of Xist expression have also been identified and
includes, protein coding genes (Rnf12) , and ncRNAs (Ftx and Jpx) [15,16] . The expression of several of these
[14]
factors is upregulated in the early stages of differentiation, explaining, at least in part, their involvement in
XCI.

The next wave of XCI is the establishment of silencing on Xi, which entails multiple steps, ensuing with the
spreading and coating of the Xist on Xi. While the active recruitment of silencing machinery to Xi is a key
component of establishing XCI, well-defined structural domains of Xist are also crucial for its function. The
structural domains of Xist could instruct the Xist:chromatin interactions and direct the subsequent assembly
of the epigenetic silencing machinery on Xi. One of the crucial regulatory region is the X inactivation center