Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells

Abstract

Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.

The function of Drosophila p53 isoforms in apoptosis

“…We first examined the protein expression levels of p53 protein isoforms by western blotting using an antibody raised against the human p53 C-terminus, which is conserved in all Drosophila isoforms. 41,42 Western blotting of wild-type larval brain and imaginal disc (B-D) extracts revealed an~48 kDa band, close to the predicted size of the 43.7 kDa p53A protein isoform, that was not detected in extracts from p53 5A-1-4 null mutants, consistent with previous results from our lab and others ( Figure 5a, lanes 1, 2). 16,41,42 The predicted p53B (55.6 kDa) and p53D/E (~36-38 kDa) isoforms were not detected, although all lanes had a non-specific 72 kDa band (Figure 5a).…”

“…42 Western blotting of extracts from the p53-Ch; p53 5A-1-4 strain with p53 antibodies revealed an abundant protein at~72 kDa, consistent with the predicted size of a p53A-mCherry fusion protein (Figure 5a).…”

“…Similar results were obtained with GAL4-driven expression in other tissues, with p53B expression always resulting in the most apoptotic cells (Table 1). 42 Overexpression of p53A or p53B induces apoptosis in ovarian follicle cells. To quantify the apoptotic potential of p53 isoforms, we overexpressed them in the adult ovary using hsp70:GAL4, a GAL4 driver that does not induce cell Figure 2 Overexpression of p53 isoforms perturbs eye development.…”

“…We previously described two fly strains transformed with a wild-type~24 kb p53 genomic BAC clone and a derivative in which mCherry was fused to the common C-terminus of all the isoforms (p53-Ch). 42,50 This BAC clone contains~17 kb 5′ and~3 kb 3′ of the p53 transcription unit and is expressed under control of the normal p53 regulatory regions, and both BACs were transformed into the same attP docking site using phiC31 transformation. Our previous results indicated that both the wild-type and the p53-Ch BAC rescued the defective apoptotic response of p53 5A-1-4 and p53…”

“…16,41 Previous evidence indicated that p53B can induce apoptosis when overexpressed, but the normal physiological function of p53B has not been evaluated. 41,42 Two shorter isoforms have also been predicted by RNA-Seq. The p53E mRNA isoform appeared in the annotated genome in the year 2014 and predicts a 334 AA (~38 kD) protein (Figures 1a and b).…”