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.

Author Summary

In order to maintain genome integrity, eukaryotic cells have evolved multiple ways to respond to DNA damage stress. One of the major cellular responses is apoptosis, during which the cell undergoes programmed cell death in order to prevent the propagation of the damaged genome to daughter cells. Although clinical observations and other studies have shown that tissues can differ in their apoptotic response, the molecular mechanisms underlying these differences are largely unknown. We have shown in our model system, Drosophila, that endocycling cells do not initiate cell death in response to DNA damage. The endocycle is a cell cycle variation that is widely found in nature and conserved from plant to animals. During the endocycle, cells duplicate their genomic DNA but do not enter mitosis to segregate chromosomes, resulting in a polyploid genome content. In this study, we investigate how the apoptotic response to DNA damage is repressed in endocycling cells. We find that the Drosophila ortholog of the human p53 tumor suppressor protein is expressed at very low levels in endocycling cells. Moreover, the downstream pro-apoptotic genes that are regulated by p53 are epigenetically silenced in endocycling cells. Our results provide important insights into tissue-specific apoptotic responses in development, with possible broader impact on understanding radiation therapy response and cancer of different tissues.