Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-␣ subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-␣ subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1␣ and HIF-2␣, with HIF-1␣ suppression manifesting substantially greater effects than HIF-2␣ suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.The response of cells to low oxygen (hypoxia) is characterized by coordinated regulation of the expression of a large number of genes whose products have widespread roles, including energy provision, vascular supply, and growth. Studies of the regulation of many such genes by oxygen have implicated a central role for the transcription factor hypoxia-inducible factor (HIF), 3 which exists as a heterodimer of an ␣ and a ␤ subunit (1). The mechanism of oxygen sensing, which controls this heterodimeric factor, has been elucidated recently (for reviews see Refs. 2 and 3).In the presence of oxygen, HIF-␣ molecules undergo ubiquitination followed by rapid proteasomal degradation. The ubiquitination is facilitated by the product of the von Hippel-Lindau gene (VHL), which acts as an essential component of an E3 ubiquitin ligase (4). In the presence of oxygen, the VHL protein recognizes and binds to two specific hydroxyproline residues in HIF-1␣ and HIF-2␣ (5-7). Three homologous 2-oxoglutarate-dependent dioxygenases PHD1, PHD2, and PHD3 catalyze this prolyl hydroxylation (8, 9). Further oxygenregulated control of the transcriptional potency of HIF-␣ is provided by another 2-oxoglutarate-dependent dioxygenase (FIH-1), which catalyzes the formation of a specific hydroxyasparagine in the C terminus of HIF-␣, decreasing its binding to the transcriptional coactivator p300 (10, 11).The identification of this mechanism of regulating HIF r...

The effects of hypoxia on gene transcription are mainly mediated by a transcription factor complex termed hypoxia-inducible factor (HIF). Genetic manipulation of animals and studies of humans with rare hereditary disease have shown that modifying the HIF pathway affects systems-level physiological responses to hypoxia. It is, however, an open question whether variations in systems-level responses to hypoxia between individuals could arise from variations within the HIF system. This study sought to determine whether variations in the responsiveness of the HIF system at the cellular level could be detected between normal individuals. Peripheral blood lymphocytes (PBL) were isolated on three separate occasions from each of 10 healthy volunteers. After exposure of PBL to eight different oxygen tensions ranging from 20% to 0.1%, the expression levels of four HIF-regulated transcripts involved in different biological pathways were measured. The profile of expression of all four transcripts in PBL was related to oxygen tension in a curvilinear manner. Double logarithmic transformation of these data resulted in a linear relationship that allowed the response to be parameterized through a gradient and intercept. Analysis of variance (ANOVA) on these parameters showed that the level of between-subject variation in the gradients of the responses that was common across all four HIF-regulated transcripts was significant (P = 0.008). We conclude that statistically significant variation within the cellular response to hypoxia can be detected between normal humans. The common nature of the variability across all four HIF-regulated genes suggests that the source of this variation resides within the HIF system itself.

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