Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF‐1α, a basic helix–loop–helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF‐1α for hypoxia‐induced growth arrest and activation of p21cip1, a key cyclin‐dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21cip1 activation has been elusive. Here we demonstrate that HIF‐1α, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1. The HIF‐1α antagonism is mediated by displacing Myc binding from p21cip1 promoter. Neither HIF‐1α transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF‐1α. In keeping with its antagonism of Myc, HIF‐1α also downregulates Myc‐activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF‐1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

Oxygen homeostasis is crucial for a myriad of developmental, physiological, and pathophysiological processes. Hypoxia-inducible factor 1␣ (HIF-1␣) plays a pivotal role in response to hypoxia by transcriptionally activating target genes involving oxygen uptake, transport, delivery, and consumption. HIF-1␣ activity is regulated primarily through the ubiquitin-proteasome degradation pathway, which targets the oxygen-dependent degradation domain (ODD) of HIF-1␣. In particular, the von Hippel-Lindau (VHL) protein complex, an E3 ubiquitin ligase, binds to the ODD upon hydroxylation of HIF-1␣ Pro-564. Here, we show that in vivo VHL interacts with the N-terminal as well as the C-terminal ODD independently, supporting the notion of functional redundancy within the ODD. Moreover, we demonstrate that Leu-574 of HIF-1␣ is essential for VHL binding to the C-terminal ODD. Despite the presence of Pro-564, deletion or mutation of Leu-574 resulted in a loss of VHL binding and a gain of protein stability. Furthermore, the identification of Leu-574 redefines the N-terminal activation domain of HIF-1␣ to be constitutively active. Taken together, this study provides new insight into the mechanisms underlying VHL-mediated HIF-1␣ degradation and transcriptional activation, and a molecular basis for drug targeting.