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Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Abstract

Two forms of X-chromosome inactivation (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) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm)1,2. Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

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Figure 1: RLIM accumulates during oocyte maturation.
Figure 2: A maternally transmitted Rnf12 deletion allele leads to early embryonic lethality specifically in females.
Figure 3: Rnf12 is not required for initiation of random XCI.
Figure 4: Regulation of Xist cloud formation and X silencing by Rnf12 during imprinted XCI.

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Acknowledgements

We thank V. Boyartchuk, T. Fazzio, E. Heard, P. Kaufman, O. Rando, D. Riethmacher and J. Sharp for advice and/or reagents, D. Kim for help in ES cell analysis, and J. Zhu for statistics. I.B. is a member of the University of Massachusetts DERC (DK32520). This work was supported by National Institutes of Health grants R01CA131158 (National Cancer Institute) and 5 P30 DK32520 (National Institute of Diabetes and Digestive and Kidney Diseases) to I.B., and GM053234 to J.B.L.

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Authors

Contributions

J.S. and I.B. conceived and designed the experiments. M.B., I.H.-B. and I.B. generated the floxed Rnf12 mice. J.S. and Y.C. established and analysed ES cell lines. J.S., M.B., H.M., M.B., N.T.-I., X.Z. and B.J. performed experiments. All authors analysed the data. I.B. wrote the manuscript.

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Correspondence to Ingolf Bach.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-14 with legends. (PDF 4592 kb)

Supplementary Movie 1

This movie shows a significant number of cells in the ICM - see Supplementary Information file for full legend. (MOV 2570 kb)

Supplementary Movie 2

This movie shows embryos develops Xist clouds - see Supplementary Information file for full legend. (MOV 6453 kb)

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Shin, J., Bossenz, M., Chung, Y. et al. Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice. Nature 467, 977–981 (2010). https://doi.org/10.1038/nature09457

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