Hypoxia-inducible Transcription Factor Binding Research Articles

The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer.

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance

BackgroundHypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia.ResultsWe report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth.ConclusionsOur data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

Integrated epigenomic profiling reveals endogenous retrovirus reactivation in renal cell carcinoma.

BackgroundTranscriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior.MethodsWe performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA).FindingsThough HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC.InterpretationIntegrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.

Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal Trichoplax adhaerens.

BackgroundIn humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens.MethodsWe report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals.ResultsHigh resolution crystal structures (≤1.3 Å) of TaPHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons.ConclusionConserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.

Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF binding to a MYC enhancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel–Lindau tumour suppressor (VHL) and unrestrained activation of hypoxia-inducible transcription factors (HIFs). Genetic and epigenetic determinants have an impact on HIF pathways. A recent genome-wide association study on renal cancer susceptibility identified single-nucleotide polymorphisms (SNPs) in an intergenic region located between the oncogenes MYC and PVT1. Here using assays of chromatin conformation, allele-specific chromatin immunoprecipitation and genome editing, we show that HIF binding to this regulatory element is necessary to trans-activate MYC and PVT1 expression specifically in cells of renal tubular origins. Moreover, we demonstrate that the risk-associated polymorphisms increase chromatin accessibility and activity as well as HIF binding to the enhancer. These findings provide further evidence that genetic variation at HIF-binding sites modulates the oncogenic transcriptional output of the VHL–HIF axis and provide a functional explanation for the disease-associated effects of SNPs in ccRCC.

Oxygen-dependent regulation of aquaporin-3 expression.

The purpose of this study was to investigate whether aquaporin-3 (AQP3) expression is altered in hypoxia and whether hypoxia-inducible transcription factor (HIF)-1 regulates the hypoxic expression. AQP3 mRNA expression was studied in L929 fibrosarcoma cells and in several tissues derived from mice that were subjected to hypoxia. Computational analysis of the AQP3 promoter revealed conserved HIF binding sites within close proximity to the translational start site, and chromatin immunoprecipitation assays confirmed binding of HIF-1α to the endogenous hypoxia response elements. Furthermore, hypoxia resulted in increased expression of AQP3 mRNA in L929 fibrosarcoma cells. Consistently, shRNA-mediated knockdown of HIF-1α greatly reduced the hypoxic induction of AQP3. In addition, mRNA analysis of organs from mice exposed to inspiratory hypoxia demonstrated pronounced hypoxia-inducible expression of AQP3 in the kidney. Overall, our findings suggest that AQP3 expression can be regulated at the transcriptional level and that AQP3 represents a novel HIF-1 target gene.

Abstract P2-05-01: The non-coding transcriptome of hypoxic breast cancer: Novel insights of clinical relevant long non-coding RNA in hypoxia signalling

Abstract Hypoxia is associated with aggressive and poor prognosis of breast cancer. Generally, pan-genome analyses of hypoxia have focussed on protein-coding genes, however, the role of non-coding RNAs, in particular long non-coding RNAs (lncRNA) in hypoxia is not well characterised. We undertook an integrated genomic analysis of the hypoxic transcriptome in MCF7 breast cancer cells, employing total RNA-seq together with ChIP-seq for the hypoxia-inducible transcription factor (HIF) and for epigenetic marks of transcriptional activation (RNApol2 and histone H3K4me3). Analyses revealed that all classes of RNA are significantly regulated by hypoxia including piwiRNA, miRNA, tRNA, and sn/snoRNA. Significant numbers of lncRNAs were upregulated in hypoxia and were associated with increased RNApol2 and H3K4me3 markers and with HIF binding, indicating direct transcriptional activation of lncRNAs by HIF. The most hypoxiclly upregulated lncRNA was NEAT1, which is a direct transcriptional target of HIF-2a but not HIF-1a. The role of NEAT1 in cancer has not been previously studied. We demonstrated that hypoxic NEAT1 induction is common in breast cancer cell lines and xenografts models treated with bevacizumab. NEAT1 directly induces the formation of nuclear paraspeckle bodies in hypoxia. Moreover, it contributes to tumourigenicity by increasing cell proliferation, colony formation, and reducing apoptosis. In addition, we report that NEAT1 is required to retain hypoxia induced hyper edited Junctional Adhesion Molecule A (JAM-A) mRNA in the nucleus, thus preventing export into the cytoplasm for translation. Finally, in a large cohort of 2000 breast cancers, high levels of NEAT1 were associated with poor clinical outcomes and clinicopathological features. Our results extend knowledge of the hypoxic transcriptional response into the spectrum of non-coding transcripts. These findings provide novel mechanisms of transcriptional regulation in hypoxia and open new avenues to find novel pathways and targets to develop therapies for breast cancer. Citation Format: Hani Choudhry, Johannes Schodel, Ashwag Albukhari, Syed Haider, Francesca Buffa, Peter J Ratcliffe, David R Mole, Ioannis Ragousis, Adrian L Harris. The non-coding transcriptome of hypoxic breast cancer: Novel insights of clinical relevant long non-coding RNA in hypoxia signalling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-01.

Unlocking the complexity of hypoxia non-coding transcriptome landscape of breast cancer

Transcriptional responses to hypoxia are central to the pathogenesis of many types of cancer. Today, pan-genome analyses of hypoxia have focused on protein-coding genes, however, the role of non-coding RNAs, in particular long non-coding RNAs (lncRNA) is not well characterised. We undertook an integrated pan-genomic analysis of the transcriptional responses to hypoxia in MCF7 breast cancer cells, employing total RNA-seq together with ChIP-seq for the hypoxia-inducible transcription factor (HIF) and for epigenetic marks of transcriptional activation (RNApol2 and histone H3K4me3). Analyses revealed that all classes of RNA are significantly regulated by hypoxia. We found significant numbers of lncRNAs are up-regulated in hypoxia and these are associated with epigenetic marks of increased transcription and HIF binding. We describe a number of hypoxia regulated non-annotated RNA species, including several that are antisense to hypoxia regulated protein-coding RNAs. The most hypoxia up-regulated lncRNA was NEAT1. The role of NEAT1 in cancer has not been previously studied. We demonstrate that NEAT1 induction is common in breast cancer cell lines and xenograft models. Finally, selected hypoxia regulated lncRNAs are analysed in a large cohort of breast cancers (n=2000) and found to be associated with poor clinical outcome and clinicopathological features. Our findings extend knowledge of the hypoxic transcriptional response into the spectrum of non-coding transcripts. These HIF-regulated non-coding transcripts have the potential to act as biomarkers for breast cancer as well as potential novel therapeutic targets.

Regulation of Epithelial Innate Immunity through Hypoxia-mediated Autophagy

The gastrointestinal epithelium comprises the primary barrier between omnipresent luminal antigens and the underlying immune cell repertoire, and is subject to profound metabolic fluctuations, particularly in inflammatory bowel disease (IBD). A role for the hypoxia-inducible transcription factors (HIFs) in orchestrating transcriptional changes to promote barrier function has been well defined. Given the importance of HIF signaling to epithelial homeostasis, we hypothesized that HIF-mediated transcriptional changes contribute to epithelial innate immunity. Promoter microarray screens of HIF-enriched genomic DNA, derived from chromatin immunoprecipitation (ChIP-on-chip), highlighted autophagy as a novel pathway coordinately regulated by HIF-1. A fundamental role for autophagy in IBD pathogenesis has recently been elucidated, defined largely by abrogated bacterial killing (xenophagy). However, little is known about transcriptional regulation of coordinated autophagy gene responses, and how these modulate intestinal epithelial cells (IEC) xenophagy. Human intestinal epithelial Caco-2 and T84 cells were used. Hypoxia was defined as pO2 20 torr and pCO2 35 torr. ChIP-on-chip: ChIP was performed with a polyclonal HIF-1a antibody using sheared chromatin from hypoxic Caco-2 cells. ChIP-enriched and input DNA were Cy-labeled and hybridized to a promoter microarray (Switchgear Genomics). The log2 ratio (input-Cy3/HIF-ChIP-Cy5) was analyzed to identify sequences specific for HIF-1a binding. ChIP-qPCR: ChIP-qPCR was used to quantify HIF binding to candidate loci using primer sets flanking putative HRE sites. Salmonella Typhimurium Invasion and O2 Consumption: Caco-2 cells were infected with late-log cultures of S. Typhimurium 14028 at an multiplicity of infection (MOI) of 100. The SDR OxoDish system (Presens) was used to measure the effect of Salmonella invasion on epithelial O2 consumption in real-time. Gentamicin kill assays: Caco-2 cells were infected with S. Typhimurium and incubated in antibiotic-free medium for 30 min, followed by media supplemented with 50 μg/mL gentamicin (Sigma) for 1h. Viable intracellular bacteria were quantified by colony counts following epithelial lysis in 1% Triton-X-PBS. A cohort of promoters for autophagy genes were specifically enriched for HIF binding by ChIP-chip. Transcript levels of candidate genes were induced by 6hrs under hypoxia or prolyl hydroxylase inhibition in Caco-2 and T84 cells, corroborating association between HIF binding and gene expression. Hypoxic Caco-2 cells displayed 3.5-fold higher LC3/p62-positive puncta by IF than normoxic cells. By immunoblot analysis, LC3-II and p62 protein levels were increased and decreased respectively in hypoxic lysates, indicating hypoxia-induced autophagic turnover. Hypoxia stimulation of autophagy increased Salmonella killing, confirming hypoxic induction of xenophagy. Moreover, invasive Salmonella induced a time- and MOI-dependent depletion of O2 with stabilization of HIF protein. To analyze baseline epithelial autophagy in the absence of HIF signaling in vivo, we measured IEC levels of LC3 and p62 protein in HIF-1b−/− mice. Consistent with a role for HIF, the ratio of LC3-I to LC3-II and total levels of p62 were increased in vil-Cre+/HIF-1bfl/fl mice relative to vil-Cre−/HIF-1bfl/fl controls. Modulation of HIF-mediated autophagy by inflammatory hypoxia and/or bacterial pathogens themselves defines a conserved innate response for host protection, and may represent a novel target for development of therapeutic strategies to modify epithelial autophagic targeting of bacteria.

Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors

BackgroundHepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.Methodology/Principal FindingsHepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.Conclusions/SignificanceTaken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.

Characterization of Tissue-Specific HIF-1 and HIF-2 Regulatory Elements of the Erythropoietin Gene

Hypoxia regulates erythropoiesis and other essential processes via hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers that consist of an α subunit (3 isotypes with significant homology; HIF-1α, HIF-2α, HIF-3α), and a common b-subunit; HIF-1 and HIF-2, in some instances exhibiting tissue- and gene-specific gene regulation. Erythropoietin (EPO) was the first identified HIF-1 target gene with the defined HIF-1 binding sequence. However, subsequent works suggested that HIF-2 also regulates EPO transcription and that there are other regulatory elements of EPO gene (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE). In silico analysis of the human EPO genome found two additional potential HIF-binding elements in the KIE and NRE regions. The comparative analysis of phylogenically conserved sequences of human, mouse, dog, and rat Epo genes further refined these mouse Epo gene HIF-binding elements as mKIE, mNRE1, mNRE2, and mNRLE2.We treated mice in hypoxia chamber (8% O2) and monitored changes of Epo mRNA levels in liver, kidney, brain, spleen, and bone marrow. All tested tissues increased Epo transcription during hypoxia. Bone marrow, spleen, kidney, and brain showed a peak of induction of Epo transcript at 3 hours of hypoxia treatment, while liver reached the highest level at 6 hours. Mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF- 2α and tissue-specific binding regions were defined. The results from these studies are summarized below.HIF-1mKIErnNREmNRE2mNRLE2NormHypNormHypNormHypNormHypLiver−+−−+−??Kidney−+−−+−+−Brain−+−−−+−+BM−+−−−−−+Splsen−+−−−−−+HIF-2mKIEmNREmNRE2mNRLE2NormHypNormHypNormHypNormHypLiver−+−−−+−+Kidney+−−−+−??Brain−−−−−−−+BM−−−−−−+−Spleen−+−−−−−+“+” denotes presence and “-” absence of binding of HIF-1 and HIF-2, “?” – indicates inconclusive results. “Norm” - normoxia, “Hyp” - hypoxia.In conclusion, we demonstrate the differential hypoxia-induced binding of HIF-1 and HIF-2 at different HIF binding elements in the tissues known to express Epo. Further studies will be required to define the function of these HIF-1 and HIF-2 binding elements in tissue specific Epo expression and their role in health and disease.

A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove

AbstractThe molecular basis of the erythrocytosis group of red cell disorders is incompletely defined. Some cases are due to dysregulation of erythropoietin (Epo) synthesis. The hypoxia inducible transcription factor (HIF) tightly regulates Epo synthesis. HIF in turn is regulated through its α subunit, which under normoxic conditions is hydroxylated on specific prolines and targeted for degradation by the von Hippel Lindau (VHL) protein. Several mutations in VHL have been reported in erythrocytosis, but only 1 mutation in the HIF prolyl hydroxylase PHD2 (prolyl hydroxylase domain protein 2) has been described. Here, we report a novel PHD2 mutation, Arg371His, which causes decreased HIF binding, HIF hydroxylase, and HIF inhibitory activities. In the tertiary structure of PHD2, Arg371 lies close to the previously described Pro317Arg mutation site. These findings substantiate PHD2 as a critical enzyme controlling HIF and therefore Epo in humans, and furthermore suggest the location of an active site groove in PHD2 that binds HIF.