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Page 10 of 15 Przanowski et al. J Transl Genet Genom 2018;2:2 I http://dx.doi.org/10.20517/jtgg.2017.03

hnRNPK is an RBP and a member of the hnRNP protein family. Another member of this family is hnRNPU,
is a Xist-interacting protein, and loss of hnRNPU reactivated several Xi-linked genes . Along with hnRNPU,
[58]
hnRNPK was one of the most abundant proteins identified in a ChIRP-MS screen carried out using cells with
doxycycline-inducible Xist on chromosome 11 . This protein was identified through multiple screens using
[47]
different cell lineages; epiblast stem cells (EpiSCs) that had just undergone random XCI and trophoblast
stem cells where Xp is always silenced . The reactivation of Xi-linked Usp9x in hnRNPK-depleted EpiSCs,
[47]
confirmed that hnRNPK regulates XCI . Surprisingly, northern blot analysis showed that the depletion
[47]
of hnRNPK does not impact Xist abundance or splicing . As with SPEN, hnRNPK depletion did not alter
[47]
Xist localization on Xi, however PRC2 recruitment was significantly decreased. Chu et al. also showed that
[47]
hnRNPK directly binds Xist, with its strongest interaction located downstream of Repeat F.
Chromatin modifiers identified through the screens
The global state of chromatin undergoes extensive remodeling, causing transition from a relatively open
configuration to a more compact state. SWI/SNF is a part of the chromatin-remodeling complex found in
all living organisms. In eukaryotes, this complex is capable of altering the position of nucleosomes along
DNA, which considerably impacts transcription . SWI/SNF is also a platform that interacts with many
[59]
transcription factors and chromatin remodeling proteins. A few components of this complex were found to
be important for XCI, including SMARCA4 and SMARCD2 . It is suggested that SWI/SNF interaction
[34]
[48]
with Xist is required for proper maintenance of PRC2 function on the Xi .
[48]
Transcriptional regulators of XCI
There are number of cis-acting regulatory elements that are located in the Xic. Several studies have used
[16]
[60]
genetic approaches to delineate their roles. Identified factors include non-coding RNA genes Jpx , Linx ,
Tsx , Xite , and Ftx as well as protein-coding genes Rnf12 , Chic1 , Ppnx , Xpr , and Nap1L2 .
[63]
[17]
[14]
[63]
[61]
[62]
[64]
[15]
With few exceptions, the exact mechanism by which these factors modulate Xist or Tsix function remains to
be determined. While interrogation of cis-regulation of XCI continues, the trans-regulation of this process
has also garnered immense interest. Trans-regulation of XCI refers to the regulation of X-linked gene
expression by diffusible factors that can either activate or inhibit the silencing of Xi. As discussed earlier,
[65]
pluripotency factors are one of the trans-acting negative regulators of XCI .
[34]
Recently, Bhatnagar et al. and Sripathy et al. have carried out unbiased large-scale genome-wide screens
[32]
to identify cis and trans-acting regulators of XCI. Both the screens used fundamentally different reporters
to identify Xi reactivators but identified several XCIFs in common [Table 1 and Figure 3]. Among identified
XCIFs there is a large representation of BMP/TGF-β signaling pathway components found in three different
studies [32,34,40] [Table 1 and Figure 3]. Inhibition of this pathway was shown to decrease Xist expression.
Interestingly, Xist promoter contains several SMAD-binding motifs in close proximity to the YY1-binding site
crucial for anchoring Xist to Xi. This suggests that several regulatory pathways may converge to stringently
regulate Xist expression. Additionally, RNF12 also controls SMAD transcription factor levels, a process that
may function as a potential feedback loop for regulating Xist levels .
[66]
Additionally, the identification of XCIFs raises the prospects of Xi reactivation as a therapeutic strategy
due to several reasons. First, several of the XCIFs identified through the screens possess enzymatic activity
that can be targeted by pharmacological agents, for instance DNMT1 and RRM2. Secondly, another major
category of XCIFs identified includes serine/threonine kinases, such as for example, PI3kinase signaling
network (PDPK1, SGK2 and PRKD3), ACVR1 receptor, DNMT1 and Aurora kinase A, which can be targeted
by several small molecule inhibitors.

The 3D conformation of the Xi
The X chromosome was originally identified as a Barr body due to its peculiar heterochromatin structure
in the nucleus. Classical cytological and electron microscopy studies have revealed a unique ultrastructure