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Evidence of Xist RNA-independent initiation of mouse imprinted X-chromosome inactivation

Abstract

XX female mammals undergo transcriptional silencing of most genes on one of their two X chromosomes to equalize X-linked gene dosage with XY males in a process referred to as X-chromosome inactivation (XCI). XCI is an example of epigenetic regulation1. Once enacted in individual cells of the early female embryo, XCI is stably transmitted such that most descendant cells maintain silencing of that X chromosome2. In eutherian mammals, XCI is thought to be triggered by the expression of the non-coding Xist RNA from the future inactive X chromosome (Xi)3,4,5; Xist RNA in turn is proposed to recruit protein complexes that bring about heterochromatinization of the Xi6,7. Here we test whether imprinted XCI, which results in preferential inactivation of the paternal X chromosome (Xp), occurs in mouse embryos inheriting an Xp lacking Xist. We find that silencing of Xp-linked genes can initiate in the absence of paternal Xist; Xist is, however, required to stabilize silencing along the Xp. Xp-linked gene silencing associated with mouse imprinted XCI, therefore, can initiate in the embryo independently of Xist RNA.

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Figure 1: Dynamics of X-linked gene expression assayed by RNA FISH in two-, four-, eight- and 16-cell WT and Xp– Xist 1lox (Mut) female mouse embryos.
Figure 2: Dynamics of X-linked gene expression assayed by allele-specific RT–PCR in WT and Xp– Xist 1lox (Mut) 8–16-cell mouse embryos and embryonic-day-6.5 extra-embryonic tissues.
Figure 3: Expression of paternal X-linked green fluorescent protein (Xp– GFP ) transgene in WT and Xp– Xist 1lox (Mut) pre- and post-implantation-stage female mouse embryos.

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Acknowledgements

Acknowledgments We thank R. Jaenisch for Xist2lox mice and G. Martin for Zp3–Cre mice (Supplementary Methods). We also thank A. Fedoriw and K. Huynh, for advice on allele-specific RT–PCR, and are grateful to S. Malpani and L. Williams, for discussions; M. Calabrese, R. Chandler, D. Ciavatta, A. Fedoriw, C. Griffin, S. Malpani, J. Mugford, M. Pohlers, K. Shpargel and L. Williams, for critically reading the manuscript. We also acknowledge the Michael Hooker Microscopy Facility at the University of North Carolina at Chapel Hill for the use of their LeicaSP2 confocal microscope. This work was supported in part by an American Cancer Society Postdoctoral Fellowship to S.K. and by grants from the US National Institutes of Health to S.K. and T.M.

Author Contributions S.K. and T.M. had devised the study and designed the experimental strategy. S.K. conducted the experiments with assistance from S.P. in mouse genotyping, RNA FISH and RT–PCR experiments and from R.B.B. in RT–PCR experiments. S.K. and T.M. analysed the data. J.S. performed the statistical evaluation of the RNA FISH and RT–PCR data. S.K. wrote the paper and T.M. edited the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Terry Magnuson.

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Kalantry, S., Purushothaman, S., Bowen, R. et al. Evidence of Xist RNA-independent initiation of mouse imprinted X-chromosome inactivation. Nature 460, 647–651 (2009). https://doi.org/10.1038/nature08161

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