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RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast

Abstract

In 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 chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome 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 RLIM (also known as Rnf12)3,4,5. Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.

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Figure 1: Downregulation of RLIM levels in early embryonic tissues before and after implantation.
Figure 2: Rlim is dispensable at post-implantation stages in female mice.
Figure 3: Detection of Xist clouds in somatic tissues of cKOmp females.
Figure 4: Normal kinetics of XCI in female mouse embryos lacking RLIM.

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Acknowledgements

We are grateful to H. Ma for help in the mouse facility, M. Keeler for culturing ES cells, T. Fazzio, N. Lawson and Y. Yoon for advice, and T. Fazzio for helpful discussions and reading of the manuscript. I.B. is a member of the University of Massachusetts Diabetes-Endocrinology Research Center (DK32520). This work was supported by National Institutes of Health grants CA131158 to I.B., CA077735 to S.N.J. and GM053234 to J.B.L.

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Authors

Contributions

J.S. and I.B. conceived and designed the experiments. M.Bo. generated the floxed Rlim mice. J.G. and S.N.J. generated the ES-cell lines and performed the tetraploid injections. M.By. and J.B.L. carried out RNA-FISH experiments and M.C.W., C.M. and J.M. performed immunohistochemistry on early embryos. All authors analysed the data. I.B. wrote the manuscript with input from J.S.

Corresponding author

Correspondence to Ingolf Bach.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Strain-independent downregulation of RLIM in the mouse embryonic epiblast.

a, Test of specificity of the RLIM antibody (compare with Fig. 1b). Shown is an E4.5 Δ/Y blastocyst outgrowth stained with RLIM (red). Increasing the general signal levels (RLIM′) reveals augmented unspecific staining mainly in the cytoplasm of cells. b, Co-stainings of sections of E7–7.5 embryos within decidual tissues using antibodies directed against RLIM (red) and H3K27me3 (green). Representative C57BL/6 and SV129 embryos are shown (out of at least three that were stained). Scale bars, 75 μm.

Extended Data Figure 2 Genetic background has little or no influence on RLIM dispensability during rXCI.

a, b, C57BL/6–SV129 (a) and C57BL/6–FVB (b) hybrid F1 parents were generated by crossings of fl/Y (C57BL/6) males with WT/WT (SV129 or FVB) females and WT/Y (SV129 or FVB) males with cKO/Δ-SC (C57BL/6) females. F1 WT/fl females and cKO/Y-SC males were then backcrossed to generate F2 cKOm offspring. Percentages of offspring (grouped into female and male) and their genotypes with respect to Rlim and SC are indicated in the abscissa and ordinate, respectively, and the total number (n) of born and genotyped F2 pups is shown. Maternally transmitted cKO alleles are indicated in black. c, No discrimination against SV129 in born cKOmp pups with a mixed C57BL/6–SV129 background. Sequencing analyses of genomic DNA isolated from eight hybrid cKOmp pups using 156 strain-specific SNPs distributed among all chromosomes (blue/red) or ten SNPs distributed on the X chromosome (green/orange). Note that the SV129 contribution in born cKOmp pups is up to 70% (total) and 80% (on the X).

Extended Data Figure 3 Co-stainings of MEFs isolated from cKOmp and WT/WT embryos using antibodies directed against H3K27me3 (green) and RLIM (red).

a, Representative images are shown. b, Summary graph of cells showing H3K27me3 foci is shown on the right. Numbers of counted cells from independent biological duplicates are 111/112 (WT/WT) and 110/104 (cKOmp).

Extended Data Figure 4 Random XCI in female mice heterozygous for the Sox2-Cre-mediated deletion of Rlim.

Female mice were generated carrying either a paternal or maternal Rlim deletion and a GFP transgene on the other X chromosome (XGFP). Mating schemes to generate these females are indicated. Female littermates without the Sox2-Cre transgene were used as controls. Numbers of cells counted from independent biological duplicates are indicated.

Extended Data Figure 5 Female ES cells lacking RLIM are able to undergo rXCI in vivo.

a, Parental cross used for the generation of ES-cell lines is indicated on top. Newly generated ES-cell lines were genotyped using PCR for the presence of wild-type, floxed or knockout Rlim alleles, as well as Cre driver (SC) and Y chromosome (Zfy2). b, Newly isolated female ES cells were differentiated for 5 days in culture and stained with the H3K27me3 antibody. c, Summary of the tetraploid complementation assays showing the injected ES-cell lines, genotypes, number of deciduas and embryos obtained (see also Fig. 4c). All embryos generated via tetraploid complementation were genotyped for the presence of wild-type, floxed and knockout Rlim alleles as well as Zfy2. d, Left, MEFs isolated from an E10.5 embryo generated via tetraploid injection of Δ/Δ line IB11 ES cells were cultured for 24 h before staining with antibodies against H3K27me3. Right, summary of H3K27me3 stainings. Numbers of counted cells from independent biological duplicates are 60/62 (WT/WT) and 78/75 (cKOmp). e, Summary of RNA-FISH experiments on MEFs isolated from embryos generated via tetraploid injection of Δ/Δ line IB11 ES cells (n = 109) (see Fig. 4d). MEFs isolated from WT/WT embryos (n = 106) served as control.

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Shin, J., Wallingford, M., Gallant, J. et al. RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast. Nature 511, 86–89 (2014). https://doi.org/10.1038/nature13286

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