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

Shin J, Bossenz M, Chung Y, Ma H, Byron M, Taniguchi-Ishigaki N, Zhu X, Jiao B, Hall LL, Green MR, Jones SN, Hermans-Borgmeyer I, Lawrence JB, Bach I - Nature (2010)

Bottom Line: 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).We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited.These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

View Article: PubMed Central - PubMed

Affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School (UMMS), Worcester, Massachusetts 01605, USA.

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). 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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A maternally transmitted Rnf12 deletion allele leads to early embryonic lethality specifically in females. Embryos were first photographed and then processed for genotyping in B–H. A)MMTV-Cre mediated loss of Δm females. Schematic diagram of born pups of indicated mating schemes (1–5). Parental genotypes of female (upper) and male (lower) mice with respect to Rnf12 and MMTV-Cre is shown and the total number (n) of F1 offsprings and the mean litter size is indicated. Number of offsprings (grouped in female and male) and their genotypes with respect to Rnf12 are indicated in the abscissa and ordinate, respectively. m (maternal) and p (paternal) indicate the origin of the KO (Δ) allele. In mating scheme 4 and 5, maternally transmitted wt, floxed and Δ alleles are indicated in grey, blue and red, respectively. Three asterisks indicate P values <1×10−7. B–E) Heterozygote Δm/fl female and homozygote Δm/Y littermates from a fl/Δp × fl/Y cross at E7.5 (A); E8.5 (B); E9.5 (C) and E10.5 (D). F) Heterozygote fl/Δp female at E10.5 from a fl/fl × Δ/Y cross. G) Best developed Δm/fl embryo (n=28) detected at E10.5 (magnification of the Δm/fl shown in F). H) Representative Δm female embryo at E10.5. Scale bars = 0.15 mm (B); 0.4 mm (C); 0.6 mm (D); 1 mm (E, F); 0.5 (G); 0,25 mm (H).
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Figure 2: A maternally transmitted Rnf12 deletion allele leads to early embryonic lethality specifically in females. Embryos were first photographed and then processed for genotyping in B–H. A)MMTV-Cre mediated loss of Δm females. Schematic diagram of born pups of indicated mating schemes (1–5). Parental genotypes of female (upper) and male (lower) mice with respect to Rnf12 and MMTV-Cre is shown and the total number (n) of F1 offsprings and the mean litter size is indicated. Number of offsprings (grouped in female and male) and their genotypes with respect to Rnf12 are indicated in the abscissa and ordinate, respectively. m (maternal) and p (paternal) indicate the origin of the KO (Δ) allele. In mating scheme 4 and 5, maternally transmitted wt, floxed and Δ alleles are indicated in grey, blue and red, respectively. Three asterisks indicate P values <1×10−7. B–E) Heterozygote Δm/fl female and homozygote Δm/Y littermates from a fl/Δp × fl/Y cross at E7.5 (A); E8.5 (B); E9.5 (C) and E10.5 (D). F) Heterozygote fl/Δp female at E10.5 from a fl/fl × Δ/Y cross. G) Best developed Δm/fl embryo (n=28) detected at E10.5 (magnification of the Δm/fl shown in F). H) Representative Δm female embryo at E10.5. Scale bars = 0.15 mm (B); 0.4 mm (C); 0.6 mm (D); 1 mm (E, F); 0.5 (G); 0,25 mm (H).

Mentions: Pups born by fl/fl females displayed normal gender ratios and a normal transmission of the paternal X chromosomes (p) was observed in matings using wt, fl or Δp males (Fig. 2A, mating schemes 1–3). However, no female offspring carrying a maternally transmitted KO (Δm) allele was born by fl/fl-Cre or wt/fl-Cre females crossed with wt, fl or Δp males (Fig. 2A, schemes 4, 5; not shown) whereas the Δm allele transmitted efficiently to male pups. The probability of obtaining male versus female pups from fl/fl-Cre or wt/fl-Cre females was highly significant (P<2 × 10−8). In contrast, wt/fl-Cre females transmitted the wt allele normally and the probability of producing male wt/Y versus male fl/Y + Δm/Y offsprings was similar (P>0.13) (scheme 5). We also performed matings with the MMTV-Cre line D that does not target to the female germline 11 and observed normal Mendelian distributions for male and females pups (n=177; not shown) sired by fl/fl-Cre (line D) females. These findings combined with a decreased mean litter size for matings 4 and 5 indicated that the deletion of Rnf12 in the maternal germ line leads to embryonic lethality. As mice were bred in a congenic C57BL/6 background to eliminate strain-specific influences, our results reveal a sex-specific parent-of-origin effect.


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

Shin J, Bossenz M, Chung Y, Ma H, Byron M, Taniguchi-Ishigaki N, Zhu X, Jiao B, Hall LL, Green MR, Jones SN, Hermans-Borgmeyer I, Lawrence JB, Bach I - Nature (2010)

A maternally transmitted Rnf12 deletion allele leads to early embryonic lethality specifically in females. Embryos were first photographed and then processed for genotyping in B–H. A)MMTV-Cre mediated loss of Δm females. Schematic diagram of born pups of indicated mating schemes (1–5). Parental genotypes of female (upper) and male (lower) mice with respect to Rnf12 and MMTV-Cre is shown and the total number (n) of F1 offsprings and the mean litter size is indicated. Number of offsprings (grouped in female and male) and their genotypes with respect to Rnf12 are indicated in the abscissa and ordinate, respectively. m (maternal) and p (paternal) indicate the origin of the KO (Δ) allele. In mating scheme 4 and 5, maternally transmitted wt, floxed and Δ alleles are indicated in grey, blue and red, respectively. Three asterisks indicate P values <1×10−7. B–E) Heterozygote Δm/fl female and homozygote Δm/Y littermates from a fl/Δp × fl/Y cross at E7.5 (A); E8.5 (B); E9.5 (C) and E10.5 (D). F) Heterozygote fl/Δp female at E10.5 from a fl/fl × Δ/Y cross. G) Best developed Δm/fl embryo (n=28) detected at E10.5 (magnification of the Δm/fl shown in F). H) Representative Δm female embryo at E10.5. Scale bars = 0.15 mm (B); 0.4 mm (C); 0.6 mm (D); 1 mm (E, F); 0.5 (G); 0,25 mm (H).
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Related In: Results  -  Collection

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Figure 2: A maternally transmitted Rnf12 deletion allele leads to early embryonic lethality specifically in females. Embryos were first photographed and then processed for genotyping in B–H. A)MMTV-Cre mediated loss of Δm females. Schematic diagram of born pups of indicated mating schemes (1–5). Parental genotypes of female (upper) and male (lower) mice with respect to Rnf12 and MMTV-Cre is shown and the total number (n) of F1 offsprings and the mean litter size is indicated. Number of offsprings (grouped in female and male) and their genotypes with respect to Rnf12 are indicated in the abscissa and ordinate, respectively. m (maternal) and p (paternal) indicate the origin of the KO (Δ) allele. In mating scheme 4 and 5, maternally transmitted wt, floxed and Δ alleles are indicated in grey, blue and red, respectively. Three asterisks indicate P values <1×10−7. B–E) Heterozygote Δm/fl female and homozygote Δm/Y littermates from a fl/Δp × fl/Y cross at E7.5 (A); E8.5 (B); E9.5 (C) and E10.5 (D). F) Heterozygote fl/Δp female at E10.5 from a fl/fl × Δ/Y cross. G) Best developed Δm/fl embryo (n=28) detected at E10.5 (magnification of the Δm/fl shown in F). H) Representative Δm female embryo at E10.5. Scale bars = 0.15 mm (B); 0.4 mm (C); 0.6 mm (D); 1 mm (E, F); 0.5 (G); 0,25 mm (H).
Mentions: Pups born by fl/fl females displayed normal gender ratios and a normal transmission of the paternal X chromosomes (p) was observed in matings using wt, fl or Δp males (Fig. 2A, mating schemes 1–3). However, no female offspring carrying a maternally transmitted KO (Δm) allele was born by fl/fl-Cre or wt/fl-Cre females crossed with wt, fl or Δp males (Fig. 2A, schemes 4, 5; not shown) whereas the Δm allele transmitted efficiently to male pups. The probability of obtaining male versus female pups from fl/fl-Cre or wt/fl-Cre females was highly significant (P<2 × 10−8). In contrast, wt/fl-Cre females transmitted the wt allele normally and the probability of producing male wt/Y versus male fl/Y + Δm/Y offsprings was similar (P>0.13) (scheme 5). We also performed matings with the MMTV-Cre line D that does not target to the female germline 11 and observed normal Mendelian distributions for male and females pups (n=177; not shown) sired by fl/fl-Cre (line D) females. These findings combined with a decreased mean litter size for matings 4 and 5 indicated that the deletion of Rnf12 in the maternal germ line leads to embryonic lethality. As mice were bred in a congenic C57BL/6 background to eliminate strain-specific influences, our results reveal a sex-specific parent-of-origin effect.

Bottom Line: 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).We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited.These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

View Article: PubMed Central - PubMed

Affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School (UMMS), Worcester, Massachusetts 01605, USA.

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). 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.

Show MeSH
Related in: MedlinePlus