Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells

Abstract

Nonhomologous DNA end-joining (NHEJ) is a major pathway of DNA double-strand break (DSB) repair in mammalian cells, and it functions to join both specifically programmed DSBs that occur in the context of V(D)J recombination during early lymphocyte development as well as general DSBs that occur in all cells. Thus, defects in NHEJ impair V(D)J recombination and lead to general genomic instability. In human patients, mutations of Cernunnos-XLF (also called NHEJ1), a recently identified NHEJ factor, underlie certain severe combined immune deficiencies associated with defective V(D)J recombination and radiosensitivity. To characterize Cernunnos-XLF function in mouse cells, we used gene-targeted mutation to delete exons 4 and 5 from both copies of the Cernunnos-XLF gene in ES cell (referred to as Cer(Delta/Delta) ES cells). Analyses of Cer(Delta/Delta) ES cells showed that they produce no readily detectable Cernunnos-XLF protein. Based on transient V(D)J recombination assays, we find that Cer(Delta/Delta) ES cells have dramatic impairments in ability to form both V(D)J coding joins and joins of their flanking recombination signal sequences (RS joins). Cer(Delta/Delta) ES cells are highly sensitive to ionizing radiation and have intrinsic DNA DSB repair defects as measured by pulse field gel electrophoresis. Finally, the Cernunnos-XLF mutations led to increased spontaneous genomic instability, including translocations. We conclude that, in mice, Cernunnos-XLF is essential for normal NHEJ-mediated repair of DNA DSBs and that Cernunnos-XLF acts as a genomic caretaker to prevent genomic instability.

Fig. 1.

Gene-targeted deletion of Cernunnos-XLF in murine ES cells. (A) The schematic diagram represents murine Cernunnos-XLF locus (Top), targeting vector (Second Row), targeted allele (Cer^N, Third Row), and the deleted allele (Cer^Δ, Bottom). The 3′ and 5′ probes are marked as black lines. The exons and loxP sites are shown as filled boxes and filled triangles respectively. Restriction site designation: B, BamHI; V, EcoRV. The map is not drawn to scale. (B) Southern blot analysis of BamHI-digested DNA from WT (lane 1), Cer^+/N(lane 2), Cer^+/Δ (lane 3), Cer^Δ/N (lane 4), and Cer^Δ/Δ (lane 5) ES cells. Lanes 6–9 represent the parent WT TC1 ES cells (lane 6), intermediate targeting (lane 7), and the homozygous Cernunnos-XLF-deficient ES cells (lanes 8 and 9) obtained through high-G418 selection and Cre deletion. The location of the 3′ probe is indicated in A. (C) Schematic representation of the human Cernunnos-XLF protein and the predicted truncated protein from Cer^Δ/Δ ES cells. Black triangles indicate the position of Cernunnos-XLF mutations identified in six human patients (two patients contain the same mutation) (5, 6). (D) Northern blot analysis of total RNA (20 μg) from WT and four different Cernunnos-XLF-deficient ES cells probed with the ORF of mouse Cernunnos-XLF. (E) RT-PCR analysis with primers flanking the entire ORF of the murine Cernunnos-XLF gene from WT or Cernunnos-XLF-deficient ES cells. (F) Western blot analyses of total protein (50 μg) from WT, Cer^N/Δ, and Cer^Δ/Δ ES cells. The predicted size for authentic Cernunnos-XLF and a faint nonspecific (NS) band are indicated with filled arrowheads.

Fig. 2.

Increased IR sensitivity and DNA double-strand-break repair defects of Cernunnos-XLF mutant ES cells. (A) IR sensitivity of WT (TC1), XRCC4^−/−, Cer^+/Δ, Cer^N//N, Cer^N/Δ, Cer^Δ/Δ, and Cer^Δ/Δ ES cells that express the WT Cernunnos-XLF cDNA from a PGK promoter (Cer^Δ/Δ + Cer). The percentage of surviving colonies in comparison to unirradiated cells is plotted as the function of irradiation dose (rad). Each data point represents the average of at least two (most times three) independent experiments performed on at least two independent ES cell clones. (B) Cernunnos-XLF deficiency causes DNA DSB repair defects. Sybr green-stained pulse-field gel electrophoresis analysis of DNA from WT (TC1), XRCC4^−/−, and two independent clones of Cer^Δ/Δ ES cells at various times after exposure to 80Gy γ-irradiation. The level of repair is inversely correlated with the amount of DNA in the gel at a given time point versus the amount of DNA that enters the gel immediately after radiation (0 h time point). See Materials and Methods and Results for other details of the assay.

Fig. 3.

Spontaneous genomic instability in Cernunnos-XLF-deficient ES cells. (A) Examples of cytogenetic abnormalities observed in untreated Cernunnos-XLF-deficient ES cell. DAPI-stained chromosomes are blue, with the centromeres being visualized as more intense blue ovals. Orange/red dots come from telomere signals. Color-coded arrowheads and corresponding cartoons indicate the different kinds of cytogenetic abnormalities that were scored (white, chromosomal break; red, chromatid break; yellow, dicentric chromosomes; green, Robertsonian translocation). One form of chromosomal break illustrated is a small piece of chromosome that contains telomeres and lacks centromeres; this anomaly is present in ≈50% of metaphases that have a chromosome that lacks telomeres on its long arms and likely represents two pieces of a broken chromosome as clearly illustrated in ref. . (B) Percentage of abnormal metaphases from WT, XRCC4^−/−, and Cernunnos-XLF-deficient ES cells quantified by telomere FISH (T-FISH) analyses. (C) Frequency of cytogenetic abnormality per metaphase in WT, XRCC4^−/−, and Cernunnos-XLF-deficient ES cells. The filled bars represent chromosomal breaks, and the open bars represent chromatid breaks. The data in B and C represents the average and standard deviation of three independent experiments from two independent WT ES cell lines, one XRCC4^−/− line, and three independent Cer^Δ/Δ or Cer^N/N ES cell lines. We note certain limitations to the T-Fish assay, which generally could result in an underestimation of chromosome breaks and translocations, in Materials and Methods.