Identification of hypoxia regulated HIF-1A and HIF-2A specific target genes in Glioblastoma
ABSTRACT: HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells. 12 samples were analysed comprising 4 experimental conditions (normoxia scr, hypoxia scr, hypoxia siHIF1, hypoxia siHIF2) in triplicate. We made pairwise comparisons between the averages of each triplicate set to normoxia scr using the Partek suite.
Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells. Overall design: 12 samples were analysed comprising 4 experimental conditions (normoxia scr, hypoxia scr, hypoxia siHIF1, hypoxia siHIF2) in triplicate. We made pairwise comparisons between the averages of each triplicate set to normoxia scr using the Partek suite.
Project description:Recently, hypoxia via the transcription factor HIF-1a has been implicated to play an important role for the fate of the adaptive immune response by regulatory T cells (Treg) and T helper 17 cells (TH17) in the mouse model. However, the reports on the effect of HIF-1a are conflicting and so far no functional data in the human system are available. Therefore, we analyzed the effect of hypoxia and HIF-1a on Treg and TH17 in the human system. FACS, western blot and reporter assays clearly demonstrated that hypoxia does not up-regulate the level of HIF-1a in CD4+ T cells (THC) and microarray analysis revealed no change of the transcriptome comparing normoxia vs. hypoxia. Furthermore, we could show that HIF-1a is almost exclusively regulated via NF-kB and NFAT, whereas hydroxylation and subsequent degradation of HIF-1a had little to no effect. In addition, we showed that HIF-1a is essential for nTreg mediated suppression and for IL-17A secretion of TH17, but not for TH17 lineage commitment measured by RORγt expression. In conclusion, our results demonstrated that THC have a distinct regulation of HIF-1a protein levels, which was absolutely essential for Treg and TH17 function. 3 patients, 2 cell type, 2 treatments = 12 arrays
Project description:Based on the results of numerous clinical and preclinical analyses, the transcription factor HIF-1a has been identified as an important tumor-promoting factor and is considered to be an attractive target for cancer therapy. To further deconstruct the molecular nature of HIF-1a’s role in tumorigenesis, we have applied lentiviral shRNA transduction to establish HIF-1a-deficient gastric cancer cells. Interestingly, functional analyses failed to show a significant growth defect of HIF-1a-deficient gastric cancer cells in vitro and in vivo. These observations led us to propose that stable inactivation of HIF-1a resulted in efficient compensation enabling cell growth and, ultimately, tumor progression in a HIF-1a-independent manner. To better understand the mechanisms that control this compensation, we performed transcriptomics of control (“scrambled” (SCR)) and HIF-1a-deficient (HIF) gastric cancer cells. Analysis of hypoxia-inducible factor-1alpha (HIF-1a)-deficient gastric cancer cells under normoxia. The transcription factor HIF-1a is a key regulator of oxygen homeostasis and has been identified as an important tumor-promoting factor. Results provide insight into the role of HIF-1a in gastric carcinogenesis. Overall design: Gastric cancer AGS cells were lentiviral and stably transduced with a small hairpin RNA targeting human HIF-1a or a control shRNA. RNA was extracted from control and HIF-1a-deficient cells. Each cell line was analyzed in triplicate for a total of six samples.
Project description:Neuroendocrine (NE) carcinoma and NE differentiation (NED) of human prostate tumor are hallmarks of aggressive human prostate cancer. Here we reveal that HIF-1a cooperation with FoxA2, a transcription factor expressed in NE tissue, is required for determining NE phenotype. Reduced HIF-1a expression, as seen in the E3 ubiquitin ligase Siah2 mutant mice, converted NE tumors to atypical hyperplasia when crossed with the TRAMPTg mice. Significantly, HIF-1a cooperation with FoxA2 enables the trans-activation of select HRE-regulated genes such as Hes6, Plod2 and Jmjd1a, whose expression is notably higher in metastatic prostate adenocarcinomas. Our findings disclose the requirement for spatial and timely regulation of FoxA2 and HIF-1a for NE/NED in prostate tumors. 12 prostate tumor samples were analyzed during normoxia and hypoxia, with different FOX/HIF genotypes. The pivotal samples are represented as duplicates.
Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-1α in colon, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-1α+/+, Hif-1αLSL/LSL mice. Background & Aims: The progression and growth of solid tumors leads to a state where tumors outgrow their capacity for efficient oxygenation and nutrient uptake and an increase in tumor hypoxia. Tumor hypoxic response is mediated by hypoxia-inducible factor (HIF)-1a and HIF-2a. These transcription factors regulate a battery of genes that are critical for tumor oxygenation, tumor metabolism, and cell proliferation and survival. Therefore, inhibitors of HIF have been sought for as anti-neoplastic agents in several different kinds of cancers. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1a is beneficial, and can reduce intestinal inflammation. The efficacy of pharmacological agents that chronically activate HIF-1a are decreased due to the tumorigenic potential of HIF. However, recent advance in understanding HIF signaling have identified mechanisms, which could allow for isoform specific activators. Activation of HIF-2a increases colon carcinogenesis and progression in mouse models. However, the role of chronic HIF-1a activation is unclear in the progression in colon cancer. The present data demonstrates that activation of HIF-1a in epithelial cells does not increase colon carcinogens or progression in two mouse models of colon cancer, and provides the proof of principle that HIF-1a activation maybe safe as therapies for inflammatory bowel disease. Global gene expression profiling in colon RNAs isolated from 6- to 8-week-old Hif-1α+/+ (n=5, Shah 019) and Hif-1αLSL/LSL (n=5, Shah 020).
Project description:Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin-remodeling, upon loss of the cardiolipin acyl transferase Tafazzin, mutes HIF-1a signaling in hypoxia. Tafazzin-deficiency does not affect posttranslational HIF-1a regulation but rather HIF-1a gene-expression, a dysfunction recapitulated in iPSCs-derived cardiomyocytes from Barth Syndrome patients with Tafazzin-deficiency. RNAseq analyses confirmed drastically altered signaling in Tafazzin mutant cells. In hypoxia, Tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-kB activation and concomitantly HIF-1a gene-expression. In agreement, Tafazzin-deficient mice hearts display reduced HIF-1a levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin-remodeling dampens HIF-1a signaling through inactivation of a non-canonical signaling pathway: Lack of NF-kB activation through reduced mitochondrial ROS production diminishes HIF-1a transcription. Overall design: Cells lacking tafazzin were subjected to hypoxia and compared to the ones at normoxia or isogenic WT cells either at hypoxia or nomoxia
Project description:Hematopoietic stem cells (HSCs), which reside in bone marrow niches, are exposed to low levels of oxygen and follow an oxygen gradient throughout their differentiation. Hypoxia-inducible factors (HIFs) are the main factors regulating the cell response to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role of HIF-1a in the maintenance of murine HSCs, however the role of HIF-2a is still unclear. Here, we show that knockdown of HIF-2a and to a much lower extent, HIF-1a impedes the long-term repopulating ability of human CD34+ umbilical cord blood derived cells. The defects observed in hematopoietic stem and progenitor cell (HSPC) function after HIF-2a knockdown was due to an increase in the production of reactive oxygen species (ROS), which increases the endoplasmic reticulum (ER) stress in HSPCs and triggers apoptosis by the activation of the unfolded-protein-response (UPR) pathway. Importantly, HIF-2a deregulation also resulted in a significant decrease of engraftment of human acute myeloid leukemia (AML) cells. Overall, our data demonstrates a key role of HIF-2a in the maintenance of human HSPCs and in the survival of primary AML cells. 2 controls and 2 shHIF2 CD34+ cell samples sorted from mice after 6 weeks of engraftment
Project description:HIF-1a and HIF-2a are expressed at high levels in mesenchymal progenitors compared to more committed mesenchymal cells and hematopoietic cells. HIF-factors could therefore have a role in the regulation the biology of mesenchymal progenitors and their functions, like the non cell-autonomous maintenance of hematopoietic progenitors. We used microarrays to detail the global program of gene expression regulated by HIF-1a or HIF-2a in mesenchymal progenitors Mesenchymal progenitors were FACS-sorted and cultured in low oxygen concentration for few days. Once cells started to form CFU-F colonies, we transduced them with shRNAs targeting specifically HIF-1a or HIF-2a. Four days after transduction, cells were collected and RNA extracted for microarray analysis.
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
Project description:Hypoxia inducible factor (HIF) is the major transcriptional regulator of cellular responses to hypoxia. The two principal HIF-a isoforms, HIF-1a and HIF-2a, are progressively stabilized in response to hypoxia and form heterodimers with HIF-1b to activate a broad range of transcriptional responses. Here we report on the pan-genomic distribution of isoform-specific HIF binding in response to hypoxia of varying severity and duration, and in response to genetic ablation of each HIF-a isoform. Our findings reveal that, despite an identical consensus recognition sequence in DNA, each HIF heterodimer loads progressively at a distinct repertoire of cell-type specific sites across the genome, with little evidence of redistribution under any of the conditions examined. Marked biases towards promoter proximal binding of HIF-1 and promoter distant binding of HIF-2 were observed under all conditions and were consistent in multiple cell type. The findings imply that each HIF isoform has an inherent property that determines its binding distribution across the genome, which might be exploited to therapeutically target the specific transcriptional output of each isoform independently. Overall design: ChIP_seq analysis of HIF-1alpha, HIF-2alpha and HIF-1beta binding under varying conditions of hypoxia and in multiple cell lines