Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor, results in early embryonic lethality and chromosomal instability - PubMed (original) (raw)

Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor, results in early embryonic lethality and chromosomal instability

Ellen E McCarthy et al. Mol Cell Biol. 2003 Jul.

Abstract

The BRCA1 tumor suppressor has been implicated in many cellular pathways, but the mechanisms by which it suppresses tumor formation are not fully understood. In vivo BRCA1 forms a heterodimeric complex with the related BARD1 protein, and its enzymatic activity as a ubiquitin ligase is largely dependent upon its interaction with BARD1. To explore the genetic relationship between BRCA1 and BARD1, we have examined the phenotype of Bard1-null mice. These mice become developmentally retarded and die between embryonic day 7.5 (E7.5) and E8.5. Embryonic lethality results from a severe impairment of cell proliferation that is not accompanied by increased apoptosis. In the absence of p53, the developmental defects associated with Bard1 deficiency are partly ameliorated, and the lethality of Bard1; p53-nullizygous mice is delayed until E9.5. This result, together with the increased chromosomal aneuploidy of Bard1 mutant cells, indicates a role for Bard1 in maintaining genomic stability. The striking similarities between the phenotypes of Bard1-null, Brca1-null, and double Bard1; Brca1-null mice provide strong genetic evidence that the developmental functions of Brca1 and Bard1 are mediated by the Brca1/Bard1 heterodimer.

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Figures

FIG. 1.

FIG. 1.

Targeted disruption of the mouse Bard1 gene. (A) A partial restriction map of the genomic region encompassing Bard1 exon 1 (Ex1; black box) is shown on top, followed by a diagram of the targeting vector used for insertion of the hygromycin resistance-EGFP gene cassette (HygEGFP) into exon 1 of the mouse Bard1 gene (bottom). A diphtheria toxin A gene (DT) was also included in the targeting construct for negative selection. The wavy lines represent plasmid sequences. Relevant restriction sites are _Eco_RI (E), _Hin_dIII (H), _Pst_I (P), and _Sal_I (S). The 5′ flanking probe used for Southern analyses, the sizes of the endogenous and expected DNA fragments, and the locations of the PCR primers a, b, and c are also shown. hyg, hygromycin resistance gene. (B) Schematics of the neomycin resistance gene (neo) targeting vector and the recombined Bard1 locus are shown. (C) Southern analysis of ES cell DNA digested with _Pst_I to confirm gene targeting. Due to an additional _Pst_I site in the hygromycin selection marker, the 7.2-kb _Pst_I fragment detected in wild-type ES cells is reduced to 5.5 kb in properly targeted ES cells. (D) Southern analysis to identify the second targeting event in heterozygous ES cells. wt, wild type. (E) Representative PCR genotyping of E6.5 embryos with primers a, b, and c. PCR amplification was conducted on embryonic DNA from wild-type (lanes 1 and 2), Bard1+/− (lane 3), and _Bard1_−/− (lane 4) embryos. The PCR products derived from wild-type and mutant alleles are 347 and 422 bp, respectively.

FIG. 2.

FIG. 2.

Development of _Bard1_−/− embryos. Hematoxylin- and eosin-stained sagittal sections of wild-type (A and E) and _Bard1_−/− (B and F) embryos at E6.5 (A and B) and E7.5 (E and F) are shown. (A) Normal (Bard1+/−) egg cylinder with proamniotic cavity (pac) and ectoplacental cone (ep). (B) Developmentally retarded Bard1 mutant embryo. (C and D) The proliferating cells of wild-type (C) and Bard1 mutant (D) E6.5 embryos are highlighted by BrdU labeling. Strong BrdU staining can be detected throughout the wild-type embryo (C), whereas the Bard1 mutant embryo has fewer BrdU-labeled nuclei (D). (E) E7.5 wild-type embryo postgastrulation with three distinct germ layers. am, amnion; y, yolk sac. (F) Bard1 homozygous mutant at the egg cylinder stage with prominent proamniotic cavity.

FIG. 3.

FIG. 3.

Impaired proliferation of _Bard1_−/− mutants in vitro. (A) Wild-type E3.5 blastocyst. (B) _Bard1_−/− E3.5 blastocysts. (C and D) Wild-type (C) and _Bard1_−/− (D) blastocyst outgrowths after 6 days in culture. Proliferation of both the ICM and the trophoblastic giant cells (TR) is evident in the wild-type embryo. In contrast, in the _Bard1_−/− mutant embryo, the ICM is completely missing and only the trophoblastic giant cells remain.

FIG. 4.

FIG. 4.

Gross morphologies of wild-type, single homozygous mutant, and double homozygous mutant embryos. (A) Normal (Bard1+/−) and mutant (_Bard1_−/−) embryos at E7.5. (B) A normal (Bard1+/−; Brca1+/−) E9.5 embryo is shown on the left. A Bard1; Brca1 (bd1/br1) double homozygous mutant embryo is shown in comparison to a single Bard1 (bd1) mutant (heterozygous for Brca1) and a single Brca1 (br1) nullizygote (wild type for Bard1) that exhibit similar extents of development. (C) A normal (Bard1+/+; _p53_−/−) E9.5 embryo on the right compared to a Bard1; p53 double homozygous mutant (_Bard1_−/−; _p53_−/−) E9.5 embryo on the left. Note that the Bard1; p53 double homozygous mutant E9.5 embryo has developed much further than any single _Bard1_−/− embryo. Scale bar, 300 μm (A) and 800 μm (B and C).

FIG. 5.

FIG. 5.

Reciprocal stability control of Bard1 and Brca1 proteins in vivo. Protein lysates of _Bard1+/+; p53_−/−, _Bard1_−/−; _p53_−/−, and _Brca1_−/−; _p53_−/− embryos were analyzed by Western blotting. Anti-NaK-ATPase was used as a loading control.

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References

    1. Abbott, D. W., M. E. Thompson, C. Robinson-Benion, G. Tomlinson, R. A. Jensen, and J. T. Holt. 1999. BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J. Biol. Chem. 274:18808-18812. - PubMed
    1. Baer, R., and T. Ludwig. 2002. The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity. Curr. Opin. Genet. Dev. 12:86-91. - PubMed
    1. Bhattacharyya, A., U. S. Ear, B. H. Koller, R. R. Weichselbaum, and D. K. Bishop. 2000. The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin. J. Biol. Chem. 275:23899-23903. - PubMed
    1. Brodie, S. G., and C. X. Deng. 2001. BRCA1-associated tumorigenesis: what have we learned from knockout mice? Trends Genet. 17:S18-S22. - PubMed
    1. Brugarolas, J., and T. Jacks. 1997. Double indemnity: p53, BRCA and cancer. p53 mutation partially rescues developmental arrest in Brca1 and Brca2 null mice, suggesting a role for familial breast cancer genes in DNA damage repair. Nat. Med. 3:721-722. - PubMed

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