Two forms of XCI ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) around the four cell stage of embryonic development. In the embryonic tissues of the inner cell mass (ICM), a random form of XCI occurs in blastocysts which inactivates either the Xp or the maternal X chromosome (Xm) 1,2. Both forms of XCI require the non-coding Xist RNA which coats the inactive X chromosome (Xi) from which it is expressed. Xist plays crucial functions for the silencing of X-linked genes including Rnf12 3,4 encoding the ubiquitin ligase RLIM. Targeting a conditional knockout (KO) of Rnf12 to oocytes where RLIM accumulates to high levels, we find that the maternal transmission of the mutant X chromosome (Δm) leads to embryonic lethality due to defective imprinted XCI. We show that in Δm female embryos the initial formation of Xist clouds and Xp silencing is inhibited. In contrast, ES cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial roles to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.
SUMMARYIn female mice, two forms of X chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp). Later, around implantation, epiblast cells of the ICM that give rise to the embryo reactivate the Xp and undergo a random form of XCI (rXCI)1,2. Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase Rnf12/RLIM3-5. While Rnf12/RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here, we show that Rnf12/RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking Rnf12/RLIM from pre-implantation stages onwards display hallmarks of XCI including Xist clouds and H3K27me3 foci and display full embryogenic potential. These results provide evidence that Rnf12/RLIM is dispensable for rXCI, indicating that in mice an Rnf12/RLIM-independent mechanism activates Xist in the embryo proper.
Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.DOI: http://dx.doi.org/10.7554/eLife.19127.001
In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), while extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12 which encodes the ubiquitin ligase Rnf12/RLIM. Here we find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy. Alveolar cells lacking Rnf12/RLIM undergo apoptosis as soon as they differentiate from mammary epithelia. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to non-random XCI in mammary epithelial cells which primarily silence their maternal X chromosomes. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and provide strong evidence for an imprinted XCI pattern in mammary epithelial cells opposite to that found in extraembryonic trophoblast cells.
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