Regulation of apoptosis in Drosophila endocycling cells

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. In this thesis, I 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. I 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, but there was no evidence for an increase in protein stability after irradiation. However, my 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. In addition, I also characterized the functions of different Drosophila p53 isoforms. My data show that the most abundant p53A isoform is necessary and sufficient to mediate the apoptotic response to DNA damage in mitotic cycling cells. Together, results from this thesis have revealed novel mechanisms by which cells in development modulate their apoptotic response, and are relevant to understanding the evolution and function of the p53 gene family in development and disease.