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Vol. 19, Issue 9, 3691-3700, September 2008

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E2F6 Inhibits Cobalt Chloride-Mimetic Hypoxia-induced Apoptosis through E2F1

Wei-Wei Yang^*, Bo Shu^*, Yi Zhu, and Huang-Tian Yang^*

*Laboratory of Molecular Cardiology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; and Department of Cardiology, Ruijin Hospital, Institute of Cardiovascular Diseases, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Submitted February 19, 2008; Revised May 13, 2008; Accepted June 9, 2008
Monitoring Editor: Kunxin Luo

E2F6, a potent transcriptional repressor, plays important roles in cell cycle regulation. However, roles of E2F6 in hypoxia-induced apoptosis are unknown. Here, we demonstrated biological functions of E2F6 in hypoxia-induced apoptosis and regulatory pathways. During hypoxia (CoCl₂, 800 µM)-induced human embryonic kidney 293 cell apoptosis, E2F6 expression was down-regulated with concurrent increases in E2F1 expression and transactivation. E2F6 overexpression abrogated hypoxia-induced apoptosis and alteration of E2F1. Conversely, specific knockdown of E2F6 by small interfering RNA had opposite effects. Chromatin immunoprecipitation assay confirmed that E2F6 regulated E2F1 expression through the transrepression of E2F1 promoter. Interestingly, E2F1 transactivation and apoptosis induced by hypoxia in cells stably expressing E2F1 were inhibited by E2F6 overexpression, suggesting that the inhibitory effects of E2F6 are not only mediated by the repression of E2F1 promoter. This was confirmed by E2F6-inhibited transactivation of E2F1 and apoptosis via competing with E2F1 for DNA binding sites evidenced by the different behaviors of E2F6C (C-terminal deletion) and E2F6.E68 (mutant DNA binding site) and by the lack of association of E2f6 with E2F1 protein. Moreover, hypoxia up-regulated expression of E2F1-responsive proapoptotic gene apoptosis protease-activating factor 1 was repressed by E2F6 overexpression. Together, these findings demonstrate a novel role of E2F6 in control of hypoxia-induced apoptosis through regulation of E2F1.

Address correspondence to: Huang-Tian Yang (htyang{at}sibs.ac.cn)

Abbreviations used: Apaf-1, apoptosis protease-activating factor 1; ChIP, chromatin immunoprecipitation; DHFR, dihydrofolate reductase; EGFP, enhanced green fluorescent protein; NS, nonspecific; PCR, polymerase chain reaction.