Concordant Regulation of Gene Expression by Hypoxia and 2-Oxoglutarate-dependent Dioxygenase Inhibition

Abstract

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...

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