How Do Endocycling Cells Block Apoptosis? Bingqing Zhang, Brian R. Calvi. Biology, Indiana University, Bloomington, IN.

   Eukaryotic cells employ multiple checkpoints to preserve genome integrity. Apoptosis, which is one type of programmed cell death, is triggered by excessive DNA damage and considered a major barrier to genome instability and cancer. An important remaining question is how cell cycle programs and checkpoints differ among cells in development. Using Drosophila melanogaster as a model system, we have found that endocycling cells, which only go through G and S phases, do not apoptose in response to DNA damage. Also unlike mitotic cycling cells, endocycling cells do not engage apoptosis after over-expression of p53, but do apoptose after over-expression of the pro-apoptotic genes. This suggests that apoptosis is repressed because p53 cannot induce transcription of its target genes at the H99 locus. In support of this, qPCR and promoter reporters indicated that H99 gene expression is repressed in endocycling cells. Chromatin immunoprecipitation (ChIP) using antibodies against modified histones demonstrated an increase in silencing marks and depletion of activating marks in the endocycling cells. In addition, initial results from our genetic screen showed that knockdown of several genes that encode epigenetic silencing proteins sensitize salivary gland endocycling cells to p53 over-expression. Recent genome annotation suggests that p53 encodes different protein isoforms. Our preliminary data implicate that different p53 protein isoforms have different abilities to induce apoptosis. We are currently using a combination of genetic and biochemical methods to further characterize the regulation and function of these p53 isoforms. This study is providing general insights into the developmental regulation of the cellular response to stress and the decision to activate the apoptotic pathway.