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Przanowski et al. J Transl Genet Genom 2018;2:2 I http://dx.doi.org/10.20517/jtgg.2017.03 Page 7 of 15

resolution maps of Xist interaction with chromatin across a developmental time course and pointed to the
preferential Xist-binding to gene-rich islands before spreading to gene-poor domains .
[51]

Novel XCI players identified through unbiased genetic and proteomic screens
Since the discovery of Xist in the early 1990s, Xist has garnered great interest, but molecular details about
its function in XCI have been limited. Coating by Xist precedes the silencing of Xi and initiates a cascade of
events that are necessary for the maintenance and establishment of the silencing. Several of these functions are
attributed to the characteristic molecular domains of Xist that include an abundance of repetitive sequences.
It has also been speculated that Xist interacts with different regulators, a few of which have been identified
by targeted RNA immunoprecipitation (RIP) assay. These regulators include ATRX, YY1, hnRNPU/SAF-A,
and PRC2. YY1 and hnRNPU facilitate the recruitment of Xist to Xi during the initiation and maintenance
phase of XCI respectively. YY1 is a transcription factor that possesses bivalent capacity to bind both DNA
[20]
and RNA, thereby functioning to dock Xist to the chromatin . The YY1 binding domain in Xist has been
mapped to the Repeat C region using targeted RIP and electrophoretic mobility shift assays (EMSA) .
[20]
Another Xist-interacting protein crucial for its localization and identified through multiple screens is a
matrix protein called hnRNPU [42,47,49] . Investigation of the hnRNPU by mutational analysis revealed
[52]
arginine-glycine-glycine (RGG) motifs essential for its interaction with Xist . ATRX, a high-affinity RNA-
binding protein (RBP) directly interacts with RepA/Xist RNA and promotes loading of PRC2 . Without
[27]
ATRX, PRC2 cannot interact with Xist or spread in cis along the X chromosome . There is no denying the
[53]
fact that use of targeted approaches have identified several key players in XCI, as outlined above; however,
this progress has been slow. Recent technological advancements have revolutionized the discovery of new
regulatory factors and provided detailed information to order them in a sequential pathway. Here, we have
summarized different XCIFs discovered in recent years, based on their function in the silencing of the
mammalian X chromosome.

Xist interacting proteins
Recently, three different groups used distinct methodologies to capture proteins that interact with Xist.
These studies used RNA-centric approaches, including ChIRP-MS , iDRiP , and RAP-MS to identify
[48]
[49]
[47]
Xist interactome. All together, ~300 proteins have been identified to interact with Xist [Figure 2]. The screens
identified several previously characterized Xist-interacting proteins, including hnRNPU, hnRNPK, AURKB,
RAD21, CTCF, SPEN, and HDAC3, but the other known XCIF, like EZH2 and ATRX were not identified.
Interestingly, the three screens lacked significant overlap between the screens that could be attributed to
technical differences; for example, the efficiency of crosslinking or to the cell type used in these studies. A
more directed functional validation across different cellular systems could further address these concerns.
In the next section, we discuss some of the protein partners for Xist that have emerged from these screens
and have been functionally validated [Table 1].

SPEN (also known as SHARP or Mint) was identified through both proteomics and genetic screens and
belongs to the Split End RNA binding protein (SPEN) family of transcriptional repressors. SPEN proteins
execute transcriptional regulatory functions via three copies of RNA recognition motifs (RRMs) and
ortholog C-terminal (SPOC) domains . SPEN is a part of multi-subunit complex that consist of NCoR/
[54]
SMRT co-repressor complex required for its interaction with HDAC3. Additionally, SPEN interacts with a
regulatory ncRNA, SRA, and this interaction modulates nuclear hormone receptor signaling . McHugh
[55]
et al. found that knockdown of NCoR/SMRT or HDAC3 abrogates Xist-mediated silencing. Further,
[49]
transcriptome analysis of X-linked genes in ESCs expressing functionally defective SPEN abrogated Xist
ability to initiate gene repression . Identical results were obtained in multiple reporter systems, and showed
[43]
an increase in the expression of X-linked genes, upon RNAi-based Spen depletion [42,47,49] . Although the
X-linked gene expression was affected by the inhibition of SPEN function, surprisingly Xist coating of Xi
[43]
was not affected . This suggested that SPEN is not crucial for Xist localization, accumulation, or spreading