Female-biased embryonic death from inflammation induced by genomic instability

Abstract

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation1,2. Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex—the DNA replicative helicase comprising MCM2 to MCM73,4—that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to ‘maleness’—XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties5. Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair6. In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4Chaos3 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone.

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Fig. 1: Female-biased embryonic lethality in MCM-depleted mice.
Fig. 2: Anti-inflammatory activity of testosterone protects male embryos from the lethality induced by genomic instability.
Fig. 3: Maternal genomic-instability genotype affects viability and placental inflammation of female embryos.
Fig. 4: Dams with intrinsic genomic instability cause increased levels of DNA damage in the placenta.

Data availability

All data that underlie the findings of this study are presented in the paper, except for RNA-seq data, which have been deposited in the GEO database under accession number GSE119710. Source data are provided with the paper for the γH2AX and MCM protein quantifications.

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Acknowledgements

We thank the Cornell Transgenic Core facility for help in producing the Fancm mutant mice, and J. Grenier from the RNA Sequencing Core. This work was supported by grants from NIH (R01 HD086609 and T32 HD057854 to J.C.S., the latter grant supporting M.D.W. and J.C.B.) and the Department of Defense (BC083376 to C.-H.C.).

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Nature thanks Björn Schumacher and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Contributions

C-H.C. made the original observations of sex skewing and performed studies with testosterone and the Sry transgene while at Cornell University. A.J.M. discovered the link to inflammation and the effect of maternal genotype, and performed all experiments related to these; he also helped write and assemble the manuscript. M.D.W. performed the X chromosome-inactivation study. J.C.B. developed the Fancm mice and analysed sex ratios. J.C.S. supervised all aspects of the work and wrote the manuscript.

Corresponding author

Correspondence to John C. Schimenti.

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Extended data figures and tables

Extended Data Fig. 1 X chromosome inactivation is not perturbed in MCM-mutant embryos.

Mouse female embryos bearing one X-chromosome-linked GFP transgene were dispersed into single cells, and examined by flow cytometry for GFP fluorescence. Control mice were female littermates with a genotype of Mcm4C3/+Mcm2+/+ or Mcm4C3/C3Mcm2+/+. The centre line represents the mean, and error bars represent the s.d. in GFP+ cells among the individual embryos (n) used. There was no significance difference (n.s) between the values by an unpaired two-tailed t-test (P = 0.926). C3/C3, Mcm4C3/C3; M2/+, Mcm2Gt/+. Source data

Extended Data Fig. 2 Placental, but not embryonic, levels of MCM are decreased in MCM mutants independent of maternal genotype, and NSAID does not rescue levels of MCM.

a, Representative western blots of protein lysates from E13.5 embryos and placentae of the indicated genotypes (top of each lane), immunolabelled with antibodies against MCM2, MCM4 and β-actin. The samples came from dams of two genotypes (indicated at the top of the panel). C3/C3, Mcm4C3/C3; C3/+, Mcm4C3/+; M2/+, Mcm2Gt/+. Note that the levels of MCM4 are particularly affected. The experiment was performed twice for each maternal genotype. For gel source data, see Supplementary Fig. 1. b, Placental levels of MCM2 and MCM4 proteins from the indicated maternal genotypes were quantified from western blots (including blots additional to those shown in a), which were imaged (see Methods) and normalized to actin and protein levels in the wild type. Each plotted point represents a single placenta. P values are from an unpaired, two-tailed t-test. Placentae corresponding to male or female Mcm4C3/C3Mcm2Gt/+ genotypes are indicated. Centre lines represent the mean, and error bars indicate s.d. c, Embryonic levels of MCM2 and MCM4 proteins were determined as in b. Each plotted point represents a single embryo. Embryos corresponding to male or female Mcm4C3/C3Mcm2Gt/+ genotypes are indicated. The results were not significant by a one-way analysis of variance (ANOVA). Source data

Extended Data Fig. 3 Sex-specific altered expression of inflammation genes in mutants.

a, Heat map of the ratio of FPKM values for key genes from top-ranking genes from the following three gene set enrichment analysis hallmarks: EMT, allograft rejection and interferon-γ response. The ratios are expressed as female:male for each of the indicated embryo and dam genotypes. Data are from RNA-seq on n = 16 placentae; n = 6 Mcm4C3/C3Mcm2Gt/+ placentae of embryos from Mcm4C3/C3 dams, n = 6 Mcm4C3/C3Mcm2Gt/+ placentae of embryos from Mcm4C3/+Mcm2Gt/+ dams and n = 4 placentae of embryos from homozygous Mcm4C3/C3 matings. Equal numbers of male and female mice were used. C3/C3, Mcm4C3/C3; C3/+, Mcm4C3/+; M2, Mcm2Gt/+. b, Maternal genotype affects the expression of inflammation genes. The ratios of female:male FPKM values of Mcm4C3/C3Mcm2Gt/+ embryos for Mcm4C3/C3 dams are plotted, compared to Mcm4C3/+Mcm2Gt/+ dams for the same gene sets as in a. The highest and lowest ranked genes are all related to inflammation responses. Source data

Extended Data Table 1 Segregation of genotypes from crosses

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McNairn, A.J., Chuang, C., Bloom, J.C. et al. Female-biased embryonic death from inflammation induced by genomic instability. Nature 567, 105–108 (2019). https://doi.org/10.1038/s41586-019-0936-6

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