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Page 6 of 15 Przanowski et al. J Transl Genet Genom 2018;2:2 I http://dx.doi.org/10.20517/jtgg.2017.03

[41]
screens also encouraged the use of more focused libraries, such as the chromatin modifiers and RNA-
binding proteins . While shRNA-based libraries have immensely accelerated the field, use of Clustered
[42]
Regularly Interspaced Short Palindromic Repeats (CRISPR)-based libraries is expected to fast-track our
understanding of the mechanisms of XCI. This is mainly because CRISPR technology has a capacity to cause
complete deletions of genes that will be more powerful in identifying XCI-specific genes and in ruling out
genes that are essential for survival.

An important criterion of successful screening strategy format is a biological phenotype that provides a
powerful readout for evaluating the reactivation of Xi. In this recombination era, there has been a surge in
the development of very useful reporter in vitro tools to study XCI, including immortalized cell lines with
[43]
Xi-linked gene tagged to reporters [34,41] and mouse embryonic stem cells . With several mouse models
available, an in vivo screen may provide more therapeutically relevant gene targets. The availability of these
tools could also facilitate phenotypic screens, which would identify factors that are selective to XCI initiation,
maintenance, or establishment.

Chemical based genetics screens
Another unbiased approach to identify X chromosome regulatory factors relies on small molecule
perturbations. While chemical screens could reveal tractable molecular targets functioning in XCI, they
could also provide an efficient tool to probe the targets’ functions and outline their mechanism-of-action.
As depicted in Figure 2, the workflow of a chemical screen resembles that of a genetic screen and relies on
a phenotypic screening to identify the small molecule(s) that gives a desired phenotype. In the case of XCI,
this phenotype is measured by the activation of a reporter gene. Recently a compound screen using ~400,000
[33]
molecules identified synergism between DNA methylation and Aurora kinase pathways for XCR . Another
screen with a smaller library of ~4300 chemical inhibitors identified RRM2 inhibitors that enhance DNA
demethylation by altering nucleotide metabolism causing XCR . With an increasing repertoire of available
[41]
small molecule libraries, chemical screening holds a great promise to complement classical genetics in
discovering the regulatory factors involved in XCI.

RNA:protein interaction coupled to proteomics screens
A mammalian genome encodes several lncRNAs that associate with proteins to execute complex structural
and regulatory functions. Xist is one of the paradigmatic lncRNAs, and deciphering its interacting regulatory
functions is central to understanding the Xist-mediated epigenetic silencing of the X chromosome. Several tools
are available to interrogate the lncRNA:protein interaction from a protein-centric view, but fewer methods
have been available with lncRNA in focus. However, evolution in the field of immobilized oligonucleotide
probes, affinity aptamers, and in vitro transcribed RNAs has allowed the capture of Xist:protein interactions
in vivo [44,45] . One such assay is an extension of comprehensive identification of RNA-binding proteins by
high-throughput sequencing (CHIRP-seq ) that uses a proteomics approach to identify the Xist-bound
[46]
proteome, called CHIRP-MS . CHIRP-seq is a an affinity-based method that generates high resolution map
[47]
of lncRNA binding sites by capturing lncRNA:chromatin complexes using tiling antisense oligos.

Another method is identification of direct RNA interacting proteins (iDRiP) that captures Xist-interacting
proteins using cDNA probes for Xist . However, to minimize the background and increase the specificity,
[48]
iDRiP method requires a cross-linking step and an additional DNAse treatment. Similarly, an RNA antisense
purification-mass spectrometry (RAP-MS) method modified previous assays and captured Xist:protein
[49]
complexes by using long antisense biotinylated DNA probes .
Additionally, new methods have been developed to interrogate the spatial organization of Xi in the nuclear
3D space and are discussed in details elsewhere . An imortant example is capture hybridization analysis
[50]
of RNA targets combined with high-throughput sequencing (CHART-seq). This approach revealed high-