HIF- and non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE33100 | GEO | 2013/02/05
SECONDARY ACCESSION(S): PRJNA149511
REPOSITORIES: GEO
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Project description:Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1 in the adaptive response, however, has not been determined. Here we investigate how HIF-1 influences hypoxic adaptation throughout Drosophila development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in dHIF mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIF and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIF in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. Fly eggs were collected onto egg caps with yeast paste . The caps were replaced after overnight incubation periods and kept at 25M-BM-:C, until mid-L2. At mid-L2, larvae were transferred to a fresh egg cap with blue yeast paste (0.3% bromophenol blue), and allowed to develop until achieving the partial clear gut L3 stage (-10 to -4 hrs prior to metamorphic onset). Appropriately staged animals were moved to fresh agar plates and allowed to age an additional 6 hours at 25M-BM-:C (normoxic treatment). Alternatively, animals were placed in an airtight Modular Incubator Chamber for 6 hours at 25M-BM-:C after a gas mixture containing 4% oxygen balanced with nitrogen was flashed into the chamber (hypoxic treatment). Mutant larvae were sorted for the absence of GFP expression using dissecting stereoscope with fluorescence at mid-L2. Microarray analyses were performed on at least three biological replicates of w1118, dHIF mutants, or dERR mutants at the partial clear gut third instar larval stage and were treated for 6 hours in normoxia or 4% O2. For each biological replicate, at least 10 larvae were collected and washed with 1M-CM-^WPBS. Larvae were placed in TRIzol (Invitrogen, Carlsbad, CA) and homogenized using VWR disposable pellet mixer. Total RNA was isolated using a TRIzol/RQ1 DNase (Promega, Madison, WI) hybrid extraction protocol. Template labelings were performed using the GeneChip 3' IVT Express Kit according to the manufacturerM-bM-^@M-^Ys specifications (Affymetrix, Santa Clara, CA). Hybridizations to Affymetrix GeneChip Drosophila Genome 2.0 arrays were performed using the manufacturers recommendations. Every chip was scanned at a high resolution by the Affymetrix GeneChipM-BM-. Scanner 3000 according to the GeneChip Expression Analysis Technical Manual procedures (Affymetrix, Santa Clara, CA).
2013-02-05 | E-GEOD-33100 | biostudies-arrayexpress
Project description:Hypoxia inducible factor-1 (HIF-1) is a central transcriptional regulator of genes associated with adaptive responses to hypoxia. NPM1 is a histone chaperone found to associate with HIF-1α in a phosphorylation dependent manner and increase its activty. The aim of this study was to find if HIF-1α and NPM1 regualate gene expression under hypoxia. Transcriptome analysis using Quant-RNA-seq after HIF-1α or NPM1 silencing under hypoxia reveals a significant number of genes, the hypoxic expression of which depends on both proteins.
2021-12-15 | GSE158890 | GEO
Project description:Intestinal epithelia exist in a uniquely dynamic oxygen tension microenvironment. Adaptive responses to hypoxia in mammalian cells are regulated largely by hypoxia inducible factor (HIF) transcriptional complexes. Functional HIF exists as an obligate alpha/beta heterodimer, comprising both a constitutive subunit (HIF-1beta), and an oxygen-labile regulatory (alpha) component. To date, three regulatory subunits have been identified, namely HIF-1alpha, HIF-2alpha, and HIF-3alpha, with the highest level of sequence homology conserved between HIF-1alpha and HIF-2alpha. Despite their concurrent expression in intestinal epithelial cells, HIF-1 and HIF-2 play non-redundant roles in the regulation of an overlapping but distinct set of gene targets. In this study, we performed ChIP-on-chip analysis of chromatin isolated from hypoxic intestinal epithelia to delineate HIF-1 and HIF-2 specific loci. Comparison of HIF-1alpha ChIP-chip and HIF-2alpha ChIP-chip to map HIF-1- and HIF-2-specific gene targets across the genome.
2013-11-05 | E-GEOD-43108 | biostudies-arrayexpress
Project description:The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. Studies of the regulation of the expression of many of these genes by oxygen has implicated a role for the heterodimeric transcription factor hypoxia inducible factor (HIF). The mechanism of oxygen sensing which controls this heterodimeric factor is via oxygen dependent prolyl and asparaginyl hydroxylation by specific 2-oxoglutarate dependent dioxygenases (PHD1, PHD2, PHD3 and FIH-1). Whilst HIF appears to have a major role in hypoxic regulation of gene expression, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. The extent to which 2-oxoglutarate dependent dioxygenases are responsible for the oxygen sensing mechanism in HIF-independent hypoxic gene regulation is also unclear. Both the prolyl and asparaginyl hydroxylases can be inhibited by dimethyloxalylglycine (DMOG). Such inhibition can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischaemic disease. We have examined the extent to which the HIF system contributes to the regulation of gene expression by hypoxia, to what extent 2-oxoglutarate dependent dioxygenase inhibitor can mimic the hypoxic response and the nature of the global transcriptional response to hypoxia. We have utilised microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high level of mimicry of the hypoxic response by use of the 2-oxoglutarate dependent dioxygenase inhibitor, dimethyloxalylglycine. We have also used microarray analysis of cells treated with small interfering RNA (siRNA) targeting HIF-1alpha and HIF-2alpha to demonstrate the differing contributions of each transcription factor to the transcriptional response to hypoxia. Candidate transcripts were confirmed using an independent microarray platform and real-time PCR. The results emphasise the critical role of the HIF system in the hypoxic response, whilst indicating the dominance of HIF-1alpha and defining genes that only respond to HIF-2alpha. Keywords: Hypoxia response, gene knockdown, chemical treatment
2005-12-01 | GSE3188 | GEO
Project description:Intestinal epithelia exist in a uniquely dynamic oxygen tension microenvironment. Adaptive responses to hypoxia in mammalian cells are regulated largely by hypoxia inducible factor (HIF) transcriptional complexes. Functional HIF exists as an obligate alpha/beta heterodimer, comprising both a constitutive subunit (HIF-1beta), and an oxygen-labile regulatory (alpha) component. To date, three regulatory subunits have been identified, namely HIF-1alpha, HIF-2alpha, and HIF-3alpha, with the highest level of sequence homology conserved between HIF-1alpha and HIF-2alpha. Despite their concurrent expression in intestinal epithelial cells, HIF-1 and HIF-2 play non-redundant roles in the regulation of an overlapping but distinct set of gene targets. In this study, we performed ChIP-on-chip analysis of chromatin isolated from hypoxic intestinal epithelia to delineate HIF-1 and HIF-2 specific loci.
2013-11-05 | GSE43108 | GEO
Project description:Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a master regulator of oxygen homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions, but pauses after 20-100 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain and the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies have revealed a critical molecular mechanism by which HIF-1 stimulates gene transcription and suggest that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.
2022-02-17 | PXD024373 | Pride
Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.
2024-05-21 | PXD037020 | Pride
Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.
2024-05-22 | PXD037115 | Pride
Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.
2024-05-21 | PXD036950 | Pride
Project description:Analysis of Huh-7 hepatocarcinoma cell line depleted of NDRG3 or HIF-1α under hypoxic condition. HIF-1α and NDRG3 have distinct functions in hypoxia responses. Results provide insight into molecular basis of HIF-independent signaling in the development and progression of hypoxic tumors Gene expression profiles of Huh-7 cells stably expressing NDRG3-shRNA or HIF-1α-shRNA under normoxia were compared to gene expression profiles of Huh-7 stable cells under hypoxia for 6, 12 and 24 hours.
2015-07-24 | E-GEOD-55212 | biostudies-arrayexpress