Ku86 represses lethal telomere deletion events in human somatic cells

Abstract

Nonhomologous end joining (NHEJ), a form of DNA double-strand break (DSB) repair, is conserved from bacteria to humans. One essential NHEJ factor is Ku, which consists of a heterodimer of Ku70 and Ku86. In a plethora of model systems, null mutations for Ku70 or Ku86 present with defects in DNA DSB repair, variable(diversity)joining [V(D)J] recombination, and/or telomere maintenance. The complete loss of Ku from bacteria to mice is, however, compatible with viability. In striking contrast, human patients with mutations of either Ku subunit have never been described. Here, we have used recombinant adeno-associated virus-mediated gene targeting to produce a human somatic cell line that expresses a conditionally null allele of Ku86. The induced loss of Ku86 results in cell death accompanied by massive telomere loss in the form of t-circles. Thus, Ku86 is an essential gene in human somatic cells because of its requirement, not in NHEJ or V(D)J recombination, but in telomere maintenance.

Fig. 1.

Scheme for functional inactivation of the human Ku86 locus. (A) A diagram of a partial Ku86 genomic locus. Exons are shown (not to scale) as numbered rectangles. In addition, a diagram of the rAAV targeting vector is shown. The filled triangles represent LoxP sites, and the hatched rectangle represents the NEO gene. (B) Diagram of the Ku86^NEO/+ cell line generated by the first round of targeting. (C) Ku86^NEO/+ cells were transiently transfected with a Cre expression plasmid (+pGK-Cre), and a clone of cells (Ku86^flox/+) that had lost the NEO gene but retained the floxed exon 3 was identified. (D) Ku86^flox/+ cells were then subjected to a second round of targeting using an rAAV exon 3 knockout vector. This generated the Ku86^flox/NEO cell line. (E) The Ku86^flox/NEO cell line was then infected with AdCre (+AdCre), and a clone of cells that were G418 sensitive (Ku86^flox/−) was isolated. (F) These Ku86^flox/− cells can be converted into Ku86 null (Ku86^−/−) cells at the investigator's discretion by infecting them with AdCre.

Fig. 2.

Characterization of a human Ku86 conditionally null cell line. (A) Documentation of the loss of Ku86 protein. Cell lines were exposed to the indicated dose of AdCre, and 3 days later whole-cell extracts were prepared and analyzed by Western blot analyses using antibodies directed against Ku86, Cre, and tubulin. A single blot is shown that was sequentially probed, stripped, and reprobed. (B) Same as A except that a time course rather than a dose–response is shown. AdCMV is a control, empty vector. (C) Molecular evidence for the loss of the floxed exon 3. The indicated cell lines were exposed to no virus, AdCMV, or AdCre virus. Genomic DNA was isolated 5 days later and subjected to PCR using primers that flank exon 3. A control PCR was performed by using a primer set located within intron 2. Both sets of PCR reactions were electrophoresed on agarose gels and stained with ethidium bromide. (D) Ku86^flox/− cells exposed to Cre stop growing. The indicated cell lines were plated out 2 days before infection (day 0). The growth of the cells was monitored by counting live (trypan blue-excluding) cells at subsequent days. (E) Ku86^flox/− cells exposed to Cre recombinase die. Ku86^flox/− cells were exposed to the indicated amounts of either AdCre or AdCMV virus and then fixed and stained with crystal violet ≈14 days later.

Fig. 3.

Human Ku86 null cells accumulate DNA DSBs. (A) Immunofluorescent detection of DNA DSBs. Ku86^flox/− cells were either left untreated (No Infection) or were infected with AdCre or AdCMV. Cells were fixed 5 days later and stained with fluorescently tagged secondary antibodies specific for γ-H2AX (green) or 53BP1 (red) primary antibodies. DNA was identified by DAPI staining (blue), and all 3 panels were then overlaid (merge). (Magnification: 100×.) (B) Ku86^flox/− cells have a higher incidence of γ-H2AX foci. A total of 100 to 200 cells from each experimental condition described in A along with Ku86^+/+ cells infected with AdCre (as a Cre control) were scored for the percentage of cells containing at least one microscopically visible γ-H2AX focus and for the number of foci per positive cell.

Fig. 4.

Many of the chromosomes in human Ku86 null cells lack telomeres. FISH analyses of metaphase chromosomes with a telomere-specific Cy3-(C₃TA₂)₃ protein–nucleic acid probe. Telomeres are seen as red dots, and metaphase chromosomes are stained blue. (A–D) Four independent metaphases observed in Ku86^flox/− cells treated with AdCre. The percentage of SFEs is shown for each metaphase. (E) An enlargement of 3 chromosomes observed in a metaphase derived from Ku86^flox/− cells treated with AdCre showing the apparent fusion of the sisters either with some residual telomere sequence (small red dots) or apparently lacking all telomeric sequence. Representative metaphases derived from: (F) Ku86^flox/− cells treated with AdCMV, (G) uninfected Ku86^flox/+ cells, or (H) Ku86^flox/+ cells treated with AdCre are also shown. (Magnification: 120×.)

Fig. 5.

Significant telomere loss in Ku86 null cells. (A) A total of 76 metaphases were scored for the percentage of SFEs, and the number of metaphases containing 0–10%, 10–20%, etc. SFEs is shown. Every metaphase had at least 20–30% SFEs, and most metaphases exhibited 70–80% SFE. (B) Ku86 null cells undergo telomere loss. The indicated cell lines were subjected to the indicated treatments and scored for SFEs. Total Chrs*, total chromosomes. The number is smaller than the theoretical total because some metaphases contained <46 distinguishable chromosomes, and only chromosomes that could be unambiguously scored were included.

Fig. 6.

Human Ku86 null cells contain elevated levels of extrachromosomal t-circles. Two-dimensional neutral/neutral gel electrophoresis analyses of AluI-restriction enzyme-digested genomic DNA from the indicated cell lines. After electrophoresis, each gel was transferred to nitrocellulose and hybridized with a ³²P-(C₃TA₂)₃ telomere-specific probe. (A) A diagram showing the expected arced migration of linear genomic DNA (finger) and the elevated arc expected for open-circular (“t-circle”) DNA (arrow). (B–F) Autoradiograms of the gels produced by using AluI-digested genomic DNA from the indicated cell lines: (B) uninfected Ku86^flox/− cells; (C) Ku86^flox/− cells treated with AdCMV; (D) Ku86^flox/+ cells treated with AdCre; (E) Ku86^flox/− cells treated with AdCre; and (F) uninfected ALT WI-38 VA13 cells. (Magnification: 1×.)