Abstract
BackgroundX-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.Methodology/Principal FindingsTo assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.Conclusions/SignificanceLoss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.
Highlights
X-chromosome inactivation achieves dosage compensation of X-linked genes between mammalian XX females and XY males
We have subjected AHA-A5-2b to further rounds of demethylation to yield a new subclone designated AHA4C1 that stably expresses both human XIST and mouse Xist. In these AHA-4C1 cells FISH analysis demonstrated that the human XIST RNA was not localized within the nucleus while mouse Xist RNA was localized (Figure 1A). 76% of cells had Xist localization and a diffuse XIST signal (n = 95), similar to numbers reported by Clemson et al when they analyzed a transiently-expressing hybrid [12]
To test whether any silencing occurred closer to the Xist gene than previously examined, or after stable longterm Xist expression, we examined the expression of 16 genes, including Chic1, Rnf12, Abcb7, and Atrx which are within 2 Mb of Xist
Summary
X-chromosome inactivation achieves dosage compensation of X-linked genes between mammalian XX females and XY males. The non-coding RNA XIST, which is expressed from the inactive X (Xi), but not the active X (Xa), is a critical initiator of X inactivation in eutheria. During X-chromosome inactivation, the Xist RNA coats the Xi, initiating gene silencing and the establishment of a heterochromatic state. This process leads to identifiable differences between the Xa and the Xi, some of which may be directly recruited by XIST/Xist, while others are likely indirect and due to the silenced nature of the chromatin. X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined
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