hEcd, A Novel Regulator of Mammary Epithelial Cell Survival
Abstract
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 (lox/lox) 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 Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2009
- Accession Number
- ADA516491
Entities
People
- Vimla Band
Organizations
- University of Nebraska Medical Center