Dominant suppressor genes of p53-induced apoptosis in Drosophila melanogaster

Abstract

One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Keywords: Drosophila; activating insertional mutagenesis; apoptosis; p53; suppression.

Fig. 1.

Structure of the DEP element and selective deletion of the UAS promoters. The 2 outward-directed UAS promoters are located at the ends of the mini-w⁺ DEP construct. The UAS promoters located at the 5′- and the 3′-ends are flanked by a pair of FRT and loxP sites, respectively. Each one of the UAS promoters together with the mini-w⁺ marker can selectively be deleted in vivo by the Cre and Flp recombinases (leaving the other UAS promoter intact) resulting ΔloxP and ΔFRT derivatives, respectively. The rectangular arrows at the UAS sites show the directions of the Gal4-induced transcription from the UAS promoters, and the triangles at the ends of the DEP construct represent the terminal repeats of the DEP element.

Fig. 2.

General features of DEP insertions and their genomic neighborhood. For the DNA sequence of the insertion site, see Supplementary Table 1. Arrows label the direction of gene transcription. Numbers below the arrows indicate the distance of the DEP insertion site in base pairs downstream from the gene's transcription start site. Asterisk denotes the distance is upstream from the gene's transcription start site. Thick arrows represent genes that are responsible for the suppression effect. The triangles represent the position of the DEP insertions. 5′FRT-UAS and 3′loxP-UAS with thick rectangular arrows mean the Gal4-activatable UAS promoter identified as the activator of the suppression of apoptosis. Dashed rectangular arrows mean that the apoptosis suppressor effect of neither UAS promoter can be determined unequivocally.

Fig. 3.

Effect of Gal4-activated suppressor gene mutants and their UAS-deleted derivatives on the p53-induced apoptotic r.e. phenotype. a) Wild-type adult eye. a′) Normal eye of GMR-Gal4/+ heterozygote. b) r.e. phenotype of the GMR-Gal4>UAS-p53. c and d) Apoptosis suppression effect of GMR-Gal4>suppr^DEP, UAS-p53 combinations. ΔloxP and ΔFRT stand for the UAS-deleted DEP mutant derivatives DEP^ΔloxP and DEP^ΔFRT, respectively (see Fig. 1). c) Suppression of r.e. phenotype is caused by 1 of the 2 UASs. d) Deletion of either one or the other UAS promoter results in the same r.e. phenotype, i.e. the apoptosis suppression effect cannot be definitely related to either UAS promoter.

Fig. 4.

Gal4-induced activity of the genes bearing the DEP insertion and the genes in the neighborhood. The columns represent the fold change of the gene expression measured in GMR-Gal4>Suppr^DEP, UAS-p53 vs GMR-Gal4>UAS-p53. For the numerical results, see Supplementary Table 3, *uninduced control: w; Polr2M^DEP105/TM3.