Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality

Abstract

The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls. Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation. Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.

FIG. 1.

Targeted deletion of Mrad9. (A) Structure of the targeting vector to make Mrad9 knockout ES cells and mice. The foundation is a genomic fragment of DNA containing Mrad9 from 129 SvEv mice. Other sequences: TK, tk; L, loxP sites, L; white boxes, Mrad9 exons 1 to 5; NEO; modified NEO; F, FRT sites. (B) Southern blot analysis of the Mrad9 gene in mouse ES cells. Alleles: +, wild type; Tar, containing targeting vector; −, deletion. (C) Western blot analysis of Mrad9 protein in mouse ES cells. (D) PCR to assess genotypes. The primer pair 5′-CCGGGTGAACCAATAAGGAA-3′ and 5′-AAGGAAGCAGGCATAGGCAG-3′ was used. Experimental details are found in the text.

FIG. 2.

Sensitivity of mouse ES cells to DNA-damaging agents. (A) Gamma rays; (B) UV; (C) hydroxyurea. Points are the means of three trials. Error bars indicate standard deviations.

FIG. 3.

Flow cytometric analysis of mouse ES cells mock treated or after exposure to 8 Gy of gamma rays in the absence or presence of nocodazole. Regions of the profiles corresponding to G₁, S, or G₂/M are delineated above the first row of graphs and defined below the set of graphs.

FIG. 4.

Flow cytometric analysis of BrdU-labeled mouse ES cells mock treated or after exposure to 8 Gy of gamma rays. Regions of the profiles corresponding to G₁, S, or G₂/M are delineated above the first row of graphs and defined below the set of graphs.

FIG. 5.

DNA synthesis in mouse ES cells, unirradiated or exposed to UV light at 20 J/m². Genotypes of cells are indicated. Bars represent the mean of three independent trials, indicating the ³H/¹⁴C ratio in irradiated versus unirradiated cells. Error bars indicate standard deviation.

FIG. 6.

Northern blot analyses to detect levels of mouse Hus1, Rad1, Rad17, and p21 RNA in mouse ES cells differing in the status of Mrad9. Samples in each lane are indicated. β-Actin serves as a loading control.

FIG. 7.

Gross morphology and histological sections of mouse embryos derived from Mrad9^+/− × Mrad9^+/− crosses. Photographs of intact or sectioned embryos from E6.5 (A and B), E7.5 (C to F), E8.5 (G to I), and E9.5 (J to L) are presented. Labeling for panel D is as follows: I, extraembryonic component of the chorion; II, allantois; III, neural ectoderm (neuroepithelium) in the primitive streak region. ^+/+, Mrad9^+/+; ^−/−, Mrad9^−/−.

FIG. 8.

TUNEL and BrdU uptake assays in mouse embryos derived from Mrad9^+/− × Mrad9^+/− crosses. Left set of panels: TUNEL assay to detect apoptotic cells in mouse embryos. (A) Mrad9^+/+, E8.5; (B) Mrad9^+/+, E9.5; (E) boxed region in panel A magnified; (F) boxed region in panel B magnified; (I) Mrad9^−/−, E8.5; (J) Mrad9^−/−, E9.5. Cells stained brown are undergoing apoptosis. Right set of panels: BrdU uptake assay to detect proliferation. (C) Mrad9^+/+, E8.5; (D) Mrad9^+/+, E9.5; (G) boxed region in panel C magnified; (H) boxed region in panel D magnified; (K) Mrad9^−/−, E8.5; (L) Mrad9^−/−, E9.5. Brown stain indicates cells that have taken up BrdU and are proliferating.