pineapple eye, a putative Drosophila E3 ligase for FOXO, is required for stem cell self-renewal in three adult stem cell types, female GSC, male GSC and ISC. Yalan Xing, Manisha Thuparani, Irina Kurtz, Hannele Ruohola-Baker. Department of Biochemistry, Institute for Stem Cell and Regenerative Medicine, University of Washington, SEATTLE, WA.

   Adult stem cells are key for regeneration. However, we need to understand the molecules that control this process to fully harness adult stem cell potential in future medicine. Drosophila serves as a good model for understanding adult stem cell regulation. Three excellent models for adult stem cells in Drosophila melanogaster have been extensively investigated: female and male germline stem cells (GSCs), and intestinal stem cells (ISCs) in fly midguts. Previous studies have revealed that these adult stem cell types share similar molecular regulatory pathways, suggesting that common regulators for adult stem cells can be unraveled. One of these shared regulatory pathways is Insulin receptor (InR) pathway. Through a loss-of-function screen, we identified pineapple eye (pie), a homologue of human E3 ubiquitin ligase G2E3, as an essential factor in female GSC maintenance and division. We now show that pie is also required for male GSCs self-renewal and ISCs proliferation. These data suggest that pie has a conserved role in maintaining pluripotency. One of the negative downstream targets of InR signaling pathway is transcription factor FOXO. Phosphorylation by Akt inhibits the nuclear localization and consequently the transcriptional activity of FOXO. In this study we demonstrated that pie regulates stem cell self-renewal through mediating FOXO on protein level, but not on transcription level. FOXO protein is upregulated in pie mutant stem cells. Further, reduction of FOXO efficiently rescues loss-of-pie caused stem cell loss and division defects. Based on these data, we propose a mechanism for pie action: pie regulates FOXO levels in Drosophila GSCs and ISCs, possibly through ubiquitination-mediated protein degradation. FOXO protein level in stem cells is a rheostat that is responsible for regulating self-renewal.