Jonathan M. Gleadle

C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

hsa-miR-210 Is Induced by Hypoxia and Is an Independent Prognostic Factor in Breast Cancer

Purpose: MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. Experimental Design: We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1α and HIF-2α, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearmans rank tests, univariate, and Cox multivariate analysis. Results: hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1α/VHL transcriptional system but not HIF-2α. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. Conclusions: We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1α– and VHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.

Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1α and −1β), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1α but not HIF-1β conferred oxygen-regulated activity. Two minimal domains within HIF-1α (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

Concordant Regulation of Gene Expression by Hypoxia and 2-Oxoglutarate-dependent Dioxygenase Inhibition THE ROLE OF HIF-1α, HIF-2α, AND OTHER PATHWAYS

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-α 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-α 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-1α and HIF-2α, with HIF-1α suppression manifesting substantially greater effects than HIF-2α 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.

Genome-wide Association of Hypoxia-inducible Factor (HIF)-1α and HIF-2α DNA Binding with Expression Profiling of Hypoxia-inducible Transcripts

Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-α subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1α or HIF-2α at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1α and HIF-2α (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1α- versus HIF-2α-binding sites revealed that whereas some loci bound HIF-1α in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2α contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1α versus HIF-2α understanding the mechanisms restricting HIF-2α activity will be of interest.

Hypoxic enhancement of exosome release by breast cancer cells

BackgroundExosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.MethodsBreast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.ResultsExposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.ConclusionsThese data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.

Regulation of Jumonji-domain-containing histone demethylases by hypoxia-inducible factor (HIF)-1alpha.

The transcription factor HIF (hypoxia-inducible factor) mediates a highly pleiotrophic response to hypoxia. Many recent studies have focused on defining the extent of this transcriptional response. In the present study we have analysed regulation by hypoxia among transcripts encoding human Fe(II)- and 2-oxoglutarate-dependent oxygenases. Our results show that many of these genes are regulated by hypoxia and define two groups of histone demethylases as new classes of hypoxia-regulated genes. Patterns of induction were consistent across a range of cell lines with JMJD1A (where JMJD is Jumonji-domain containing) and JMJD2B demonstrating robust, and JMJD2C more modest, up-regulation by hypoxia. Functional genetic and chromatin immunoprecipitation studies demonstrated the importance of HIF-1alpha in mediating these responses. Given the importance of histone methylation status in defining patterns of gene expression under different physiological and pathophysiological conditions, these findings predict a role for the HIF system in epigenetic regulation.

Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

ABSTRACT In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-α and HIF-β homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

Genetic Analysis of the Role of the Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-inducible Factor (HIF) in Regulating HIF Transcriptional Target Genes

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that directs a broad range of cellular responses to hypoxia. Recent studies have defined a set of 2-oxoglutarate and Fe(II)-dependent dioxygenases that modify HIF-α subunits by prolyl and asparaginyl hydroxylation. These processes potentially provide a dual system of control, down-regulating both HIF-α stability and transcriptional activity. Although genetic analyses in both primitive organisms and mammalian cells have demonstrated a critical role for the prolyl hydroxylase pathway in the regulation of HIF, analogous studies have not been performed on the HIF asparaginyl hydroxylase pathway, and its role in directing the expression of endogenous HIF transcriptional targets has not yet been clearly defined. Here we demonstrate, using small interfering RNA-mediated FIH suppression and controlled overexpression by a doxycycline-inducible system, that alterations in FIH expression in both directions have reciprocal effects on the expression of a range of HIF target genes. These effects were observed in normoxic and severely hypoxic cells but not anoxic cells. Evidence for FIH activity in severely hypoxic cells contrasted with results for the prolyl hydroxylase PHD2, suggesting that these enzymes display different oxygen dependence in vivo, with PHD2 requiring higher levels of oxygen for biological activity. Our results demonstrate an important physiological role for FIH in regulating HIF-dependent target genes over a wide range of oxygen tensions and indicate that inhibition of FIH has the potential to augment HIF target gene expression even in severe hypoxia.

Genetic Analysis of Pathways Regulated by the von Hippel-Lindau Tumor Suppressor in Caenorhabditis elegans

The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated α subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1–independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1–independent function of VHL-1 that is connected with extracellular matrix function.