Abstract-The cell cycle factor E2F-1 is known to regulate a variety of cellular processes including apoptosis. Previously we showed that disruption of Rb-E2F-1 complexes provoked apoptosis of postmitotic adult and neonatal ventricular myocytes; however, the underlying mechanism was undetermined. In this report, we show that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. Furthermore, we show mechanistically that the hypoxia-inducible death factor Bnip3 is a direct transcriptional target of E2F-1 that is necessary and sufficient for E2F-1-induced cell death. Expression of E2F-1 resulted in a 4.9-fold increase (PϽ0.001) in nucleosomal DNA fragmentation and cell death by Hoechst 33258 dye and vital staining. E2F-1 provoked mitochondrial perturbations that were consistent with permeability transition pore opening. As determined by quantitative real-time PCR analysis, a 6.2-fold increase (PϽ0.001) in endogenous Bnip3 gene transcription was observed in cells expressing wild-type E2F-1 but not in cells expressing a mutation of E2F-1 defective for DNA binding. Rb, the principle regulator of cellular E2F-1 activity, was proteolytically cleaved and inactivated in ventricular myocytes during hypoxia. Consistent with the proteolytic cleavage of Rb, chromatin immunoprecipitation analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia, a finding concordant with the induction of Bnip3 gene transcription. The Bnip3 homolog Nix/Bnip3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. 4,5 However, the signaling pathways and transcriptional processes that regulate Bnip3 transcription remain poorly defined.The cellular factor E2F-1 is the archetypal member of a family of transcription factors first characterized for their ability to activate genes required for G 1 exit and DNA synthesis. 6 To date, at least 6 homologs of E2F (E2F-1 to E2F-6), together with their dimerization partners DRFT1-polypeptides 1 and 2 (DP-1 and DP-2), have been identified. 7,8 Notably, E2F-1 is uniquely distinguished from other E2F proteins by its propensity to provoke apoptosis, 7-9 yet the underlying mechanism for this property of E2F-1 is, at best, poorly understood. In cells, E2F-1 interacts with the retinoblastoma gene product Rb via its L-X-C-X-E motif, which is crucial for suppressing cellular E2F activity and E2F-1-dependent promoters. 10 Accordingly, loss-of-function mutations or germ line deletion of Rb in mice results in increased E2F-1 activity and apoptosis. 11 Furthermore, the unexpected and counterintuitive tumorigenesis in E2F-1 Ϫ/Ϫ mice underscores the importance of E2F-1 as a key regulator of apoptosis. 12 Previously, we have shown that displacement of E2F-1 from Rb by viral oncoproteins or overexpression of E2F-1 alone was sufficient to provoke apoptosis of ventricular Original received September 21, 2007; revision received November 7, 2007; accepted December 12, 2007. myocytes; however, the underlying mechanism(s) was...
The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor Bnip3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern Bnip3 gene activity remain cryptic. Herein we show that the transcription factor NF-B provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show
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