A proximal conserved repeat in theXistgene is essential as a genomic element for X-inactivation in mouse

Abstract

X-inactivation in female mammals is triggered by the association of non-coding Xist RNA in cis with the X chromosome. Although it has been suggested that the A-repeat located in the proximal part of the Xist RNA is required for chromosomal silencing in ES cells, its role in mouse has not yet been addressed. Here, we deleted the A-repeat in mouse and studied its effects on X-inactivation during embryogenesis. The deletion, when paternally transmitted, caused a failure of imprinted X-inactivation in the extraembryonic tissues, demonstrating the essential role of the A-repeat in X-inactivation in the mouse embryo. Unexpectedly, the failure of X-inactivation was caused by a lack of Xist RNA rather than by a defect in the silencing function of the mutated RNA, which we expected to be expressed from the mutated X. Interestingly, the normally silent paternal copy of Tsix, which is an antisense negative regulator of Xist, was ectopically activated in the preimplantation embryo. Furthermore, CpG sites in the promoter region of paternal Xist, which are essentially unmethylated in the extraembryonic tissues of the wild-type female embryo, acquire a significant level of methylation on the mutated paternal X. These findings demonstrate that the DNA sequence deleted on the mutated X, most probably the A-repeat, is essential as a genomic element for the appropriate transcriptional regulation of the Xist/Tsix loci and subsequent X-inactivation in the mouse embryo.

Defects in dosage compensation impact global gene regulation in the mouse trophoblast

“…Since our previous study revealed that a simple deletion of the A-repeat abolished Xist upregulation during embryonic development (Hoki et al, 2009), we decided instead to express Xist RNA lacking the A-repeat using a CAG promoter, using the same approach as we used previously to create the Xist CAG allele (Amakawa et al, 2015). The 5′ region of Xist spanning from its endogenous promoter to the XhoI site 0.9 kb downstream of the major transcription start site in exon 1 was replaced with a fragment containing the CAG promoter and a floxed selection marker by gene targeting to generate the Xist CAGΔ5′-2L allele (Fig.…”

“…It has been suggested that this apparent discrepancy is reconciled by the finding that X-linked genes that fail to be silenced are located at the outside or periphery of the cloud formed by the Xist RNA lacking the A-repeat, and are therefore not incorporated into the apparent heterochromatin domain (Chaumeil et al, 2006), although the molecular basis of how the A-repeat exerts its effect on this spatiotemporal regulation is not known. When we previously attempted to explore this issue by targeted deletion of the A-repeat in mouse, it completely abolished the expression of Xist, precluding evaluation of the RNA lacking the A-repeat (Hoki et al, 2009).…”

Advances in understanding chromosome silencing by the long non-coding RNA Xist

“…The importance of the A-repeat in the endogenous locus has been examined in the mouse. A mutant allele, Xist DA , was produced by gene targeting [37]. Males and females hemizygous and heterozygous, respectively, for Xist DA , which is maternal in origin, were produced but intercrosses between those males hemizygous for Xist DA and wild-type females revealed that the majority of female offspring, all of which should have inherited the Xist DA allele, were lost soon after implantation.…”

Fifty years of X-inactivation research

“…MEETING REVIEW Development 138 (23) One interesting feature of Xist discussed at this meeting concerned the highly conserved A-repeat region located in the proximal part of the transcript. Previous work from Anton Wutz had shown that this region is necessary for Xist RNA to establish gene silencing during XCI (Wutz et al, 2002), and the Sado laboratory had reported that this region acts as a positive regulatory element for Xist expression (Hoki et al, 2009). This region has also been reported to produce a short independent transcript called RepA (Zhao et al, 2008).…”