Abstract
SummaryHypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.
Highlights
Hypoxia-inducible transcription factors (HIFs) are central to the metazoan hypoxia response and are key mediators of glycolysis, regulating approximately 600 genes to promote cell survival in low oxygen tensions (Benita et al, 2009; Manalo et al, 2005)
A KBM7 Forward Genetic Screen Identifies oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS) as Regulators of HIFa Stability We first developed a sensitive HIF1a reporter for use in the KBM7 forward genetic screen that would reflect the kinetics of endogenous HIF1a stability
A fluorescent construct, consisting of GFP fused with amino acids 530–603 of the HIF1a oxygen-dependent degradation domain (ODD), was expressed under the control of a minimal HIF-responsive element (HRE) promoter, forming a reporter (HIF1a-GFPODD) that could be induced and degraded in a HIF1a-dependent manner (Figure 1A)
Summary
Hypoxia-inducible transcription factors (HIFs) are central to the metazoan hypoxia response and are key mediators of glycolysis, regulating approximately 600 genes to promote cell survival in low oxygen tensions (Benita et al, 2009; Manalo et al, 2005) Undesirable consequences of their activation can promote tumor formation, lead to the development of pulmonary hypertension, and result in altered immune responses (Kaelin, 2008; Maxwell and Ratcliffe, 2002). HIF levels are likely to be regulated by a complex interplay between oxygen and metabolic signals, which are not fully understood
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