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. 2003 Apr 15;100(8):4696-701.
doi: 10.1073/pnas.0736384100. Epub 2003 Apr 2.

Drosophila p53 preserves genomic stability by regulating cell death

Naoko Sogame  1 , Misoo Kim, John M Abrams
Affiliations

Drosophila p53 preserves genomic stability by regulating cell death

Naoko Sogame et al. Proc Natl Acad Sci U S A. .

Abstract

When animal cells are exposed to stressful conditions, the tumor suppressor protein p53 restrains growth by promoting an arrested cell cycle or initiating a cell death program. How these distinct fates are specified through the action of a single protein is not known. To study its functions in vivo we produced a targeted mutation at the Drosophila p53 (Dmp53) locus. We show that Dmp53 is required for damage-induced apoptosis but not for cell-cycle arrest. Dmp53 function is also required for damage-induced transcription of two tightly linked cell death activators, reaper and sickle. When challenged by ionizing radiation, Dmp53 mutants exhibit radiosensitivity and genomic instability. Hence, elevated mutant loads were not caused by defective checkpoint functions but instead correlated with failures in p53-associated cell death. Our studies support the notion that core ancestral functions of the p53 gene family are intimately coupled to cell death as an adaptive response to maintain genomic stability.

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Figures

Figure 1
Figure 1
Generation and verification of a targeted Dmp53 mutation. (A) Targeting scheme for the Dmp53 gene. The donor construct illustrated next to a schematic of the native Dmp53 locus was generated by the insertion of a 2.8-kb internal fragment of the Dmp53 gene into the targeting vector pTV2. The 2.8-kb fragment lacks part of an N-terminal transactivation domain (because of the elimination of the entire exon 1 and part of exon 2), and part of a central DNA-binding domain and an entire tetramerization domain (because of the elimination of part of exon 7 and the entire exon 8). A germ-line transformant donor strain bearing this construct on the second chromosome was used for targeted mutagenesis as described (36, 37). FRT, FLP recombination target. On heat-shock-mediated induction of FLP site-specific recombinase and I-SceI endonuclease, the donor construct is excised as an extrachromosomal molecule and may create a lesion at the native Dmp53 locus by homologous recombination. The targeted mutation at the native Dmp53 locus, referred to as Dmp53−ns, produced a tandem duplication of Dmp53 variants that sandwich the whs gene. The dotted line in exon 6 shown for the targeted event at the Dmp53 native locus indicates an unexpected insertion of partial pTV2 vector sequences (confirmed by PCR and sequencing). (B) PCR analysis of a Dmp53-targeting event. Genomic DNA from flies was used as template DNA for PCR to verify the targeting event at the Dmp53 locus. Lanes 1–3 use a primer pair, 1 and a. Lanes 4–6 use a primer pair, 2 and b, to verify the disruption of the N-terminal Dmp53 template. Lanes 7–9 use a primer pair, 3 and c, to assess the disruption at the C-terminal Dmp53 template. All PCR fragments were partially or fully sequenced to determine the variations introduced at the native Dmp53 locus. Locations of each primer are listed in A in italics. Genotype of flies is as follows: yw refers to the parental wild-type strain at the native Dmp53 site on the third chromosome; Donor refers to the Dmp53-donor construct on the second chromosome, wild-type at the native Dmp53 site on the third chromosome; and p53−/− refers to the disrupted native Dmp53 site on the third chromosome. (C) Northern blot analysis of a Dmp53-targeting event. Total RNA from wild-type (yw) and Dmp53−ns strains was isolated, blotted to a membrane, and hybridized with a Dmp53 probe. The arrow indicates the expected 1.6-kb Dmp53 transcript. In wild-type larval and pupae individuals, this transcript is moderately responsive to ionizing radiation. Dmp53 mutants lack the1.6-kb Dmp53 transcript, and instead express aberrantly sized transcripts, which are indicated by arrowheads. (Lower) The membrane stained with methylene blue to show ribosomal RNA as a loading control. RNA from untreated (−) or γ-irradiated (+) individuals in the wandering third-instar larvae (L), pupae (P), and adult (A) stages were analyzed.
Figure 2
Figure 2
The radiation-responsive enhancer at the rpr locus is no longer radiation responsive in Dmp53−ns embryos. Transgenic lines carrying a 150-bp radiation-responsive enhancer from the rpr locus was tested in wild-type (A and B) or Dmp53−ns mutant embryos (C and D). Mock-treated controls (A and C) or γ-irradiated (B and D) samples were stained for β-galactosidase activity (25).
Figure 3
Figure 3
Dmp53−ns mutants are defective in damage-induced apoptosis. A vital dye, acridine orange, specifically recognizes apoptotic cells. Wing discs from wild-type (A and B) or Dmp53−ns (C and D) were dissected from mock-treated (A and C) or γ-irradiation-treated (B and D) larvae, and apoptotic cells were visualized by acridine orange. For each genotype, at least five wing discs were dissected and stained. The Dmp53−ns phenotype was fully penetrant.
Figure 4
Figure 4
Dmp53−ns mutants exhibit a normal cell-cycle arrest. Wing discs from wild-type (A and B) or Dmp53−ns (C and D) were dissected from mock (A and C) or γ-irradiated (B and D) third-instar wandering larvae. Histochemical staining with phosphohistone H3 antibody detects cells undergoing mitosis. For each genotype, approximately five wing discs were dissected and stained. Discs from wild-type or Dmp53−ns mutant were indistinguishable with respect to the incidence of immunoreactive cells, and staining seen for the Dmp53−ns control wing disc shown is within the normal range. The Dmp53−ns phenotype was fully penetrant.
Figure 5
Figure 5
Dmp53−ns adults carry high mutation loads after exposure to ionizing radiation. LOH is one measure of genomic instability. Here, the recessive mwh phenotype (A) was scored in flies heterozygous for mwh and either wild-type or homozygous for Dmp53−ns. (A) A wing from homozygous mwh fly. Each cell contains multiple hairs. (B) A wing from a fly heterozygous for mwh and homozygous for Dmp53−ns that has been treated with γ-irradiation. Cells within the circles have lost the wild-type copy of mwh, uncovering the recessive mwh phenotype. (C) Loss of Dmp53 leads to genomic instability after irradiation. Both mwh and Dmp53 genes are on the third chromosome, and three independent recombinant lines (mwh(1), p53; mwh(2), p53; and mwh(3), p53) were generated and tested. For each genotype, three wings were examined. The average number of mwh cells per wing in wild-type or Dmp53−ns mutant flies is indicated in flies that had or had not been exposed to moderate levels of ionizing radiation.
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