Abstract
At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.
Highlights
At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix
The introduction of a poly(A) signal in Spen−/−:Tsix-Stop lines fully rescued Xist upregulation upon differentiation (Fig. 6c), indicating that SPEN is required for Xist upregulation via Tsix repression
In this study, we show that SPEN-defective embryonic stem cells (ESCs) do not upregulate Xist upon differentiation and study the molecular mechanism behind this observation
Summary
At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. SPEN is crucial for X-linked gene silencing[16,17,19,20] by binding the Xist repeat A (RepA) via its RRM domains[17,19] and interacting via its SPOC domain with the corepressors NCoR/SMRT to recruit/activate histone deacetylase 3 (HDAC3), responsible for the removal of histone H3 and H4 acetylation at promoters and enhancers of genes located on the future Xi16,25 Despite this crucial role for SPEN in establishment of the Xi, these studies did not report defects in Xist upregulation and coating[16,17,19,20,25], possibly due to forced Xist upregulation using doxycyclineinducible systems (Supplementary Table 1). Our results indicate that SPEN is necessary for X-linked gene silencing, and plays a crucial earlier role in the regulation of initiation of XCI
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