hEcd, A Novel Regulator of Mammary Epithelial Cell Survival

Using the Yeast Two hybrid analysis with human papilloma virus oncogene E6 the most efficient oncogene to immortalize hMECs in vitro as a bait and mammary epithelial cell cDNA library, we identified hEcd human orthologue of Drosophila Ecdysoneless as a novel E6 binding partner. To study the cellular function of Ecd in mammalian cells, we generated Ecd loxlox mouse embryonic fibroblast cells from Ecd floxed mouse embryos mice generation was not supported by DOD grant . We observed that Cre-mediated deletion of Ecd in Ecdloxlox mouse embryonic fibroblasts led to a proliferative block due to a delay in G1-S cell cycle progression this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb, demonstrating Ecd plays a role in cell cycle progression via the Rb-E2F pathway. Studies in yeast suggested a potential role of Ecd in transcription however Ecd lacks a DNA binding domain. Using a GAL4-luciferase reporter assay and a GAL4-DNA binding domain DBD fusion with Ecd or its mutants, we present evidence that human Ecd has a transactivation activity in its C-terminal region. We further demonstrate that Ecd interacts with p300, a histone acetyltransferase, and the co-expression of Ecd with p300 enhances the Ecd-mediated transactivation activity. These results demonstrate human Ecd regulates 1 cell cycle progression and 2 transactivation probably by interacting with a transcriptional factor. Future studies should discover potential partners of Ecd in this process.