Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

Abstract

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory

“…SAF-A and CIZ1 are similar in that their ability to support Xist-mediated gene silencing and recruitment of Xist to Xi, respectively, is dependent on Xist E repeats (Hasegawa et al 2010), possibly identifying a common mechanism. Moreover, deletion of the E repeats was shown recently to result in dispersed localization of Xist RNA (Yamada et al 2015). These findings were attributed to SAF-A; however, our results suggest that loss of interaction with CIZ1 might contribute to the observed phenotype.…”

“…Silencing activity maps in large part to the A repeat, a short tandemly repeated region located at the 5 ′ end of Xist. In contrast, localization maps to several redundantly acting elements, including the tandem repeat regions C, E, and F (Wutz et al 2002;Jeon and Lee 2011;Makhlouf et al 2014;Yamada et al 2015).…”

SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells

“…Several tandem repeat regions (labeled A-F in the mouse) show moderate conservation (5)(6)(7), and at least two of these, repeat A and the rodent-specific repeat C, are implicated in silencing and localization to the inactive X. Deletion of the final 7.5-kb exon of Xist causes a defect in its localization (8), and the 1.5-kb region encompassing repeats F and B is required for accumulation of heterochromatic marks over the inactive X (4); however, beyond these initial characterizations, the mechanisms by which gene silencing, heterochromatinization, and localization of Xist on the X chromosome occur are not well understood. In particular, the role of RNA structure in orchestrating these distinct functions remains unclear.…”

The “lnc” between 3D chromatin structure and X chromosome inactivation

“…It appears that the first ~6kb of the transcript are sufficient to recapitulate the initiation of XCI [57]. Similar findings were obtained in a separate study that made use of female ESCs lacking the Xist terminal exon (exon 7) [79]. Whereas wild type Xist RNA is 17.9kb in length, differentiation of this mutant line resulted in expression of a stable ~10.2kb transcript.…”

“…SAF-A mutants lacking either of these nucleic acid binding domains are incapable of rescuing the Xist RNA phenotype that is observed upon depletion of SAF-A, indicating that both DNA and RNA binding functions are required to localize Xist RNA to chromatin [75]. A recently published CLIP-PCR map of the SAF-A binding sites on Xist RNA suggests that SAF-A can bind across the entire Xist transcript [79]. The location, size and density of SAF-A binding sites on chromatin remain unknown, but defining them will be important in furthering mechanistic understanding of how SAF-A regulates the chromatin-association of Xist transcripts, in particular, and lncRNAs more generally [80].…”