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. 2018 Nov 12;9(1):4752.
doi: 10.1038/s41467-018-07060-w.

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

Cristina Gontan  1 , Hegias Mira-Bontenbal  1 , Aristea Magaraki  1 , Catherine Dupont  1 , Tahsin Stefan Barakat  1 , Eveline Rentmeester  1 , Jeroen Demmers  2 , Joost Gribnau  3
Affiliations

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

Cristina Gontan et al. Nat Commun. .

Abstract

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Abrogation of rXCI in Rnf12−/− differentiating ESCs is rescued by knockout of Rex1. a Scatter plot showing the correlation of the H:L log2 ratios for the quantitatively identified proteins in the SILAC experiment in ESCs between two biological replicates (heavy-Rnf12−/−:light-WT and heavy-WT:light-Rnf12−/−) showing RNF12-dependent changes in REX1 stability. Upregulated proteins are indicated in blue (log2 ratios >0.585 and log2 ratios <−0.585 on the x and y axes, respectively). Depleted proteins are indicated in green (log2 ratios <−0.585 and log2 ratios >0.585 on the x and y axes, respectively). b Genotyping analysis of Rnf12 and Rex1 deletions in WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs. Bottom panel shows a Pf1M1 restriction fragment length polymorphism (RFLP) analysis to detect the presence of two X chromosomes in the same ESC lines. Location of green, purple and red genotyping primers is depicted in Supplementary Fig. 2b. c Nuclear extracts of WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10a. d Immunohistochemistry of REX1 (grey), RNF12 (red) and DNA (DAPI, blue) of WT, Rnf12−/− and Rnf12CR−/CR− ESCs. Note the recognition of the remaining 333 aa in RNF12−/− ESCs by the RNF12 antibody. Scale bar: 20 μm. e Xist RNA-FISH (FITC) analysis on WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs at day 6 of differentiation. DNA was stained with DAPI (blue). Scale bar: 20 μm. f Quantification of cells with Xist clouds in WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs at day 3 and day 6 of differentiation. g QPCR analysis of Xist expression in undifferentiated, day 3 and day 6 differentiated WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs (average expression ± s.d., n = 3 biological replicates)
Fig. 2
Fig. 2
Rex1 knockout mice are viable and fertile. a BAC targeting strategy to generate the Rex1 knockout cas allele in 129:cas ESCs. The Rex1 coding sequence is shown as a blue box. The start site is indicated by a black arrow. LoxP sites, denoted as red triangles, flank the neomycin-resistance gene (neo) as a positive gene selection marker. Primers used to validate gene recombination in ESCs and to genotype are shown as red arrows. b Validation of Rex1 knockout cas allele recombination by XmnI RFLP analysis of WT and Rex1-targeted ESCs (top panel). Pf1M1 RFLP analysis to detect presence of two X chromosomes (bottom panel). c Nuclear extracts of WT, Rex1+/− and Rex1−/− ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10b. d Sex and genotype distribution from different matings of Rex1-deficient mice. Number of breedings, number of mice per breeding and total number of mice are indicated below. No significant bias against the birth of female animals was observed (χ2 test, p > 0.05). e Xist RNA-FISH (FITC) analysis on WT, Rex1+/− and Rex1−/− ESCs at day 3 of differentiation. DNA was stained with DAPI (blue). White arrows indicate the presence of two clouds within a nucleus. Scale bar: 20 μm. f Quantification of Xist-positive cells (left panel) and Xist-positive cells with two clouds (right panel) in WT, Rex1+/− and Rex1−/− ESCs at day 0 and day 3 of differentiation. Asterisks indicate p-value <0.05, two-tailed Student's t test (average expression ± s.d., n = 1–3 biological replicates)
Fig. 3
Fig. 3
XCR and imprinted and random XCI in female Rex1−/− mice are not compromised. a Representative Z-stack projections of WT and Rex1−/− E3.5 and E4.5 female blastocysts immunostained for H3K27me3 (Xi marker, green), the lineage markers OCT4 (E3.5 ICM, red, left panels) and KLF4 (E4.5 epiblast, red, right panels) and DNA (DAPI, blue). Whole embryo and ICM/epiblast higher magnification for each embryo are shown (white boxes). Arrowheads mark the Xi in OCT4+ cells. KLF4+ cells display XCR (stars). Scale bars: 20 μm. b Quantification of H3K27me3 domains (green) in E3.5 and E4.5 ICMs of WT and Rex1−/− embryos in a. c Representative paraffin sections of female WT and Rex1−/− E9.5 embryo hindguts and E11.5 embryo trunks immunostained for OCT4 (PGC marker, red) and H3K27me3 (Xi marker, green). H3K27me3 domains are present in somatic cells and in some E9.5 PGCs, while they are lost in E11.5 PGCs (XCR). Representative PGCs are marked with yellow dashed lines. Scale bars: 5 μm. d Quantification of cells with an H3K27me3 domain in female WT and Rex1−/− PGCs at E9.5 and E11.5. Number of embryos and PGCs analysed are indicated. e Xist, G6pdx and Mecp2 allele-specific RNA expression analysis in E11.5 WT and Rex1−/− female embryos (E) and corresponding VYSE. LP, length polymorphism. f Quantification of the average allelic Xist (green), G6pdx (blue) and Mecp2 (pink) expression in WT and Rex1−/− embryos and VYSE in e. Light/dark colours indicate cas or 129 allelic origin, respectively. The number of mice analysed is indicated. g Representative Xist, G6pdx and Mecp2 allele-specific RNA expression analysis in heart (H), liver (Li), stomach (St), brain (Br) and lung (Lu) of WT, Rex1+/+ (from a heterozygous cross), Rex1+/− and Rex1−/− 4-week-old mice. h Quantification of the average allelic Xist (green), G6pdx (blue) and Mecp2 (pink) expression in several WT, Rex1+/+ (from a heterozygous cross), Rex1+/− and Rex1−/− 4-week-old mice in g and additional mice. Light/dark colours indicate cas or 129 allelic origin, respectively. The number of mice analysed is indicated
Fig. 4
Fig. 4
Rnf12 KO embryos show REX1 stabilization in embryonic and extraembryonic tissues. a Sex and genotype distribution from different matings of Rnf12-deficient mice in a C57BL/6 background. Number of breedings, number of mice per breeding and total number of mice are indicated. Note that no female embryos were born with a maternally transmitted Rnf12 deleted allele. No significant differences were observed between the number of females with a paternal mutant allele and their WT brothers (χ2 test, p > 0.05). A significant slight lethality associated with the mutation in male mice compared to WT brothers was observed (χ2 test, p = 1.05E−4). b Representative Z-stack projections of WT, Rnf12−/− and Rnf12−/y E4.5 blastocysts immunostained for REX1 (red), H3K27me3 (Xi marker, green), the trophectoderm marker CDX2 (grey) and DNA (DAPI, blue). Scale bars: 20 μm
Fig. 5
Fig. 5
Rnf12−/−:Rex1−/− double knockout mice are viable and have normal iXCI and rXCI. a Sex and genotype distribution from different Rnf12 mutant crossings in an Rex1-deficient background. Number of breedings, number of mice per breeding and total number of mice are indicated. Note that female embryos were born with a maternally transmitted Rnf12 deleted allele in an Rex1−/−background. No significant bias in gender or genotype was observed (χ2, p > 0.05). b Representative Z-stack projections of WT, Rnf12−/−:Rex1−/− and Rnf12−/y:Rex1−/y E4.5 blastocysts immunostained for REX1 (red), H3K27me3 (Xi marker, green), the trophectoderm marker CDX2 (grey) and DNA (DAPI, blue). Scale bars: 20 μm. WT samples are same control samples as in Fig. 4b. c Xist, G6pdx and Mecp2 allele-specific RNA expression analysis in E11.5 female Rnf12−/+:Rex1−/− embryos (E) and corresponding VYSE. d Quantification of the average allelic Xist (green), G6pdx (blue) and Mecp2 (pink) expression in E11.5 female WT and Rnf12−/+:Rex1−/− embryos and VYSE in c. Light/dark colours indicate cas or 129 allelic origin, respectively. WT samples are same control samples as in Fig. 3f. The number of mice analysed is indicated. e Xist, G6pdx and Mecp2 allele-specific RNA expression analyses in heart (H), liver (Li), stomach (St), brain (Br) and lung (Lu) in two Rnf12−/−:Rex1−/− 4-week-old female mice. f Quantification of the average allelic Xist (green), G6pdx (blue) and Mecp2 (pink) expression in WT and Rnf12−/+:Rex1−/− and Rnf12−/−:Rex1−/− 4-week-old female mice in e. Light/dark colours indicate cas or 129 origin, respectively. WT samples are the same control samples as in Fig. 3h. The number of mice analysed is indicated
Fig. 6
Fig. 6
Model for iXCI regulation by the Rex1-Rnf12 axis. In a WT background, REX1 is expressed during the early stages after fertilization, but is degraded by RNF12. This leads to the upregulation of the paternal Xist allele, whilst its maternal counterpart is prevented from upregulation by an imprint. In Rnf12−/+ embryos, the RNF12 level is sufficient to prevent REX1 stabilization, allowing for paternal Xist upregulation. However, this will result in inactivation of the paternal Rnf12 copy, which leads to an RNF12 KO situation, in its turn resulting in brief REX1 stabilization and preventing further paternal Xist upregulation. This feedback loop will prevent proper iXCI in trophoblast cells leading to Rnf12−/+ embryo death. In Rnf12−/− embryos, in the absence of RNF12, REX1 accumulates, preventing upregulation of Xist from the paternal allele. iXCI is absent and the embryos die. In Rnf12−/−:Rex1−/− embryos, the entire Rex1-Rnf12 axis is absent and iXCI can proceed normally as in WT embryos
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References

    1. Lyon MF. Gene action in the X-chromosome of the mouse (Mus musculus L.) Nature. 1961;190:372–373. doi: 10.1038/190372a0. - DOI - PubMed
    1. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E. Epigenetic dynamics of imprinted X inactivation during early mouse development. Science. 2004;303:644–649. doi: 10.1126/science.1092727. - DOI - PubMed
    1. Takagi N, Sasaki M. Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature. 1975;256:640–642. doi: 10.1038/256640a0. - DOI - PubMed
    1. Mak W, et al. Reactivation of the paternal X chromosome in early mouse embryos. Science. 2004;303:666–669. doi: 10.1126/science.1092674. - DOI - PubMed
    1. Monk M, McLaren A. X-chromosome activity in foetal germ cells of the mouse. J. Embryol. Exp. Morphol. 1981;63:75–84. - PubMed

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