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...

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.