Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-X_L Levels

Abstract

The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1^i/i) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1^i/i mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1^i/i MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X_L mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1^i/i MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We are grateful to Gerry Melino (Università di Roma Tor Vergata) for discussion and advice, Daniela Talarico (S. Raffaele Scientific Institute) and Elena Longobardi (IFOM) for critical discussions and technical help, Silvia Mori (S. Raffaele Scientific Institute) for the pBabe-Prep1 retroviral vector, and Marco Giorgio and Raluca Marcu (IFOM) for their help in JC-1 staining experiments.

This work was supported by grants from the Italian Association for Cancer Research (AIRC) (number 4/2005) Telethon and the Italian Ministry of University and Scientific Research to F.B. and from AIRC (number 63/2006) and the Istituto Superiore di Sanità to M.P.C.