Figure 2. Overview of the different proteins and REs involved in
Xist and
Tsix regulation. Genes that have an activating direct or indirect role on
Xist are shown in green, while genes involved in
Xist repression are shown in magenta. Several TF, other proteins, XCI activators, and RE are shown in green or magenta based on their effect on
Xist expression. (
A) In pluripotency, the pluripotency factor network (OCT4, NANOG, SOX2, REX1, PRDM14) keeps
Tsix active and represses
Xist, either directly by binding to Intron 1 or its promoter, or indirectly through
Tsix or by inhibiting the
Xist activator RNF12. CTCF and KAP1 also work towards inhibiting its expression, while the MSL complex supports
Tsix expression from its promoter. Another set of activating proteins, such as CHD8 and RIF1, supports low levels of
Xist expression. (
B) At the onset of XCI, reduced pluripotency factor concentrations lead to decreased
Tsix expression. Reduction in REX1 is further aided by increased RNF12 expression due to the disappearance of its pluripotent repressors. SPEN is required to shut down
Tsix expression to allow
Xist upregulation. YY1, the paralog of REX1, binds to the P2 promoter of
Xist, activating it. KDM5C and KDM6A also seem to bind there, leading to the demethylation of H3K4me2/3 to H3K4me1, a mark of enhancers, while seemingly removing H3K27me3. CHD8, however, seems to have an opposing role at the onset of XCI during differentiation because it decreases YY1s accessibility to
Xist’s promoter. CHD8 seems to fine-tune
Xist expression depending on the developmental context. Several of the GATA binding factors are required for
Xist expression during differentiation by binding several of its regulatory sequences. Shades of magenta show
Xist repressors, while shades of green indicate
Xist activators and XCI activators.
3. Distant Regulatory Regions
While the promoter regions of
Xist drive its expression, distant DNA regulatory elements (RE) seem to play a role in titrating its expression. A regulatory region located ∼10 kb downstream of
Xist TSS, and thus within
Xist intron 1, was shown to bind several pluripotency factors and play a role in
Xist repression in the pluripotent state (
Figure 2A)
[22]. A subsequent study confirmed these findings through genetic deletions of
Xist intron 1. Transgenic male ESCs carrying an
Xist intron 1 deletion show moderately upregulated
Xist expression in the pluripotent state, which is exacerbated upon
Tsix co-removal
[23]. However, in two contrasting studies, deletion of
Xist intron 1 in female ES cells does not seem to impact
Xist expression both in vivo and in vitro but rather skews the future Xi choice towards the mutated allele
[24,25][24][25].
Identification of REs involved in
Xist expression has proved to be a challenging task due to numerous factors influencing
Xist expression within the Xic. Distinguishing direct from indirect effects on
Xist expression via genetic perturbations within the Xic is difficult. However, a recent publication has successfully addressed this challenge. Using an elegant screening approach taking advantage of dCas9 fused to the repressor KRAB (CRISPRi) and detecting
Xist levels by fluorescently activated cell sorting, several candidate regions were epigenetically and systematically silenced
[26]. As expected, epigenetic inactivation of the previously discussed
Xist promoter regions, as well as of known
Xist regulators, impairs
Xist upregulation upon exit of pluripotency, serving as a validation of the screening approach. A novel regulatory cluster located ~150 kb upstream of the
Xist TSS, termed RE93-97, was also identified. Targeting this cluster with CRISPRi revealed a gradual decrease in
Xist expression, pointing to RE93-97’s role as a
Xist enhancer cluster. Furthermore, capture Hi-C analysis revealed RE93-97 interacts with
Xist’s P2 promoter upon exit of pluripotency, suggesting this regulatory cluster responds to differentiation cues.
In a follow-up study by the same group, another pivotal RE responsible for driving
Xist expression was identified
[27]. Employing a CRISPR activation screening approach, several protein factors that result in
Xist expression upon their overexpression were identified. A comprehensive analysis of potential candidates and their binding sites revealed that the GATA family of transcription factors binds not only RE93-97 but also a newly discovered regulatory element located ~100 kb upstream of the
Xist TSS termed RE79. Genetic deletions of RE97, as well as RE79, validated previous discoveries regarding the significance of RE93-97 in rXCI while elucidating the role of RE79 in driving
Xist upregulation expression in iXCI.
Gene transcription regulation is achieved not only by many different protein factors binding to promoters and REs, such as RNA Pol II, transcription factors (TFs), chromatin remodellers, etc., but also through complex chromatin architecture.