RESUMEN
The histone H3K27 demethylase, UTX/KDM6A, plays a critical role in the early development of vertebrates, and mutations are frequently found in various cancers. Several studies on developmental and cancer biology have focused on preferential transcriptional regulation by UTX independently of its H3K27 demethylase catalytic activity. Here, we analysed gene expression profiles of wild-type (WT) UTX and a catalytic activity-defective mutant in 786-O and HCT116 cells and confirmed that catalytic activity-dependent and -independent regulation contributes to the expression of most of the target genes. Indeed, the catalytic activity-defective mutant indeed suppressed colony formation similar to the WT in our assay system. However, the expression of several genes was significantly dependent on the catalytic activity of UTX in a cell type-specific manner, which could account for the inherent variation in the transcriptional landscape of various cancer types. The promoter/enhancer regions of the catalytic activity-dependent genes identified here were found to be preferentially modified with H3K4me1 and less with H3K27me3 than those of the independent genes. These findings, combined with previous reports, highlight not only the understanding of determinants for the catalytic activity dependency but also the development and application of pharmaceutical agents targeting the H3K27 or H3K4 modifications.
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Histona Demetilasas , Histonas , Neoplasias , Humanos , Dominio Catalítico , Metilación de ADN , Genes Supresores de Tumor , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Neoplasias/genética , Línea Celular TumoralRESUMEN
Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.
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Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , Poli(ADP-Ribosa) Polimerasa-1 , Hipoxia de la Célula , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Receptor Notch3/metabolismoRESUMEN
UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.
Asunto(s)
Factor de Transcripción Activador 4/agonistas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , eIF-2 Quinasa/metabolismo , Animales , Apoptosis , Benzazepinas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Unión Proteica , Pirimidinas/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.
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Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Histona Demetilasas/genética , Proteínas Nucleares/genética , Repeticiones de Tetratricopéptidos/genética , Factores de Transcripción/genética , Sustitución de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Mutación/genéticaRESUMEN
How hypoxia regulates gene expression in the human carotid body (CB) remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the impact of important post-transcriptional regulators, such as non-coding RNAs, and in particular miRNAs is not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins.
Asunto(s)
Cuerpo Carotídeo/fisiología , Hipoxia , MicroARNs/genética , Regulación de la Expresión Génica , Humanos , Técnicas In VitroRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Hypoxia-inducible factors (HIFs) facilitate cellular adaptation to environmental stress such as low oxygen conditions (hypoxia) and consequently promote tumor growth. While HIF-1α functions in cancer progression have been increasingly recognized, the contribution of HIF-2α remains widely unclear despite accumulating reports showing its overexpression in cancer cells. Here, we report that HIF-2α up-regulates the expression of CD70, a cancer-related surface antigen that improves anchorage-independent growth in cancer cells and is associated with poor clinical prognosis, which can be induced via epigenetic modifications mediated by DNMT1. The ablation of CD70 by RNAi led to decreased colony forming efficiency in soft agar. Most strikingly, we identified the emergence of CD70-expressing cells derived from CD70-negative cell lines upon prolonged hypoxia exposure or DNMT1 inhibition, both of which significantly reduced CpG-nucleotide methylations within CD70 promoter region. Interestingly, DNMT1 expression was decreased under hypoxia, which was rescued by HIF-2α knockdown. In addition, the expression of CD70 and colony forming efficiency in soft agar were decreased by knockdown of HIF-2α. These findings indicate that CD70 expression and an aggressive phenotype of cancer cells is driven under hypoxic conditions and mediated by HIF-2α functions and epigenetic modifications. This provides additional insights into the role of HIF-2α in coordinated regulation of stem-like functions and epigenetics that are important for cancer progression and may present additional targets for the development of novel combinatorial therapeutics.
RESUMEN
Hypoxia causes dramatic changes in gene expression profiles, and the mechanism of hypoxia-inducible transcription has been analyzed for use as a model system of stress-inducible gene regulation. In this study, changes in chromatin organization in promoters of hypoxia-inducible genes were investigated during hypoxia-reoxygenation conditions. Most of the hypoxia-inducible gene promoters were hypersensitive to DNase I under both normal and hypoxic conditions, and our data indicate an immediate recruitment of transcription factors under hypoxic conditions. In some of the hypoxia-inducible promoters, nucleosome-free DNA regions (NFRs) were established in parallel with hypoxia-induced transcription. We also show that the hypoxia-inducible formation of NFRs requires that hypoxia-inducible transcription factors (HIFs) bind to the promoters together with the transcriptional coactivator CBP. Within 1â¯h after the hypoxia exposure was ended (reoxygenation), HIF complexes were dissociated from the promoter regions. Within 24â¯h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible NFRs. Nucleosome reassembly required the function of the transcriptional coregulator SIN3A. Thus, reversible changes in nucleosome organization mediated by transcription factors are notable features of stress-inducible gene regulation.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/fisiopatología , Neuroblastoma/genética , Nucleosomas/fisiología , Regiones Promotoras Genéticas , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Células Tumorales CultivadasRESUMEN
Tumor hypoxia contributes to a biologically aggressive phenotype and therapeutic resistance. Recent studies have revealed that hypoxia reduces expression of several DNA damage recognition and repair (DRR) genes via both hypoxia-inducible factor (HIF)-independent and -dependent pathways, and this induced genomic instability in cancer cells. We show here that one of the HIF-target genes-differentiated embryo chondrocyte (DEC)-plays a role in DNA damage response via transcriptional repression. Comprehensive gene expression and database analyses have revealed systemic repression of DNA-DRR genes in cancer and non-cancer cells under hypoxic conditions. Hypoxic repression in typical cases was confirmed by quantitative RT-PCR and promoter reporter experiments, and knockdown experiments indicated the critical role of DEC2 in such repression. Assessment of histone H2AX phosphorylation revealed that recognition and repair of DNA double-strand breaks (DSBs) induced by bleomycin or γ-ray irradiation were attenuated; moreover, Cleaved Caspase-3 levels were decreased with pre-conditioning under hypoxia: opposing phenomena were ascertained by knockdown of DEC2. Finally, pre-conditioning under hypoxia decreased the sensitivity of cancer cells to DSBs, and knockdown of DEC2 increased γ-ray sensitivity. These data imply that a critical reduction of DNA-DRR occurs via DEC-dependent transcriptional repression and suggest that DEC is a potential molecular target for anti-cancer strategies.
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Cartílago/embriología , Hipoxia de la Célula , Condrocitos/citología , Daño del ADN , Regulación de la Expresión Génica , Transcripción Genética , Bleomicina/farmacología , Cartílago/citología , Línea Celular Tumoral , Regulación hacia Abajo , Rayos gamma , Humanos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Epigenetic mechanisms play important roles in the regulation of tumorigenesis, and hypoxia-induced epigenetic changes may be critical for the adaptation of cancer cells to the hypoxic microenvironment of solid tumors. Previously, we showed that loss-of-function of the hypoxia-regulated H3K9 methyltransferase G9A attenuates tumor growth. However, the mechanisms by which blockade of G9A leads to a tumor suppressive effect remain poorly understood. We show that G9A is highly expressed in breast cancer and is associated with poor patient prognosis, where it may function as a potent oncogenic driver. In agreement with this, G9A inhibition by the small molecule inhibitor, BIX-01294, leads to increased cell death and impaired cell migration, cell cycle and anchorage-independent growth. Interestingly, whole transcriptome analysis revealed that genes involved in diverse cancer cell functions become hypoxia-responsive upon G9A inhibition. This was accompanied by the upregulation of the hypoxia inducible factors HIF1α and HIF2α during BIX-01294 treatment even in normoxia that may facilitate the tumor suppressive effects of BIX-01294. HIF inhibition was able to reverse some of the transcriptional changes induced by BIX-01294 in hypoxia, indicating that the HIFs may be important drivers of these derepressed target genes. Therefore, we show that G9A is a key mediator of oncogenic processes in breast cancer cells and G9A inhibition by BIX-01294 can successfully attenuate oncogenicity even in hypoxia.
Asunto(s)
Hipoxia de la Célula , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Oncogenes , Transducción de Señal , Apoptosis/efectos de los fármacos , Azepinas/farmacología , Ciclo Celular , Movimiento Celular , Proliferación Celular , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células MCF-7 , Metilación , Neoplasias/enzimología , Neoplasias/patología , Pronóstico , Quinazolinas/farmacologíaRESUMEN
Hypoxia-inducible factors (HIFs) play a central role in the transcriptional response to changes in oxygen availability. Stability of HIFs is regulated by multi-step reactions including recognition by the von Hippel-Lindau tumour suppressor protein (pVHL) in association with an E3 ligase complex. Here we show that pVHL physically interacts with fatty acid synthase (FASN), displacing the E3 ubiquitin ligase complex. This results in HIF-α protein stabilization and activation of HIF target genes even in normoxia such as during adipocyte differentiation. 25-hydroxycholesterol (25-OH), an inhibitor of FASN expression, also inhibited HIF target gene expression in cultured cells and in mouse liver. Clinically, FASN is frequently upregulated in a broad variety of cancers and has been reported to have an oncogenic function. We found that upregulation of FASN correlated with induction of many HIF target genes, notably in a malignant subtype of prostate tumours. Therefore, pVHL-FASN interaction plays a regulatory role for HIFs and their target gene expression.
RESUMEN
Adaptation to hypoxia, a hallmark feature of many tumors, is an important driver of cancer cell survival, proliferation and the development of resistance to chemotherapy. Hypoxia-induced stabilization of hypoxia-inducible factors (HIFs) leads to transcriptional activation of a network of hypoxia target genes involved in angiogenesis, cell growth, glycolysis, DNA damage repair and apoptosis. Although the transcriptional targets of hypoxia have been characterized, the alternative splicing of transcripts that occurs during hypoxia and the roles they play in oncogenesis are much less understood. To identify and quantify hypoxia-induced alternative splicing events in human cancer cells, we performed whole transcriptome RNA-Seq in breast cancer cells that are known to provide robust transcriptional response to hypoxia. We found 2005 and 1684 alternative splicing events including intron retention, exon skipping and alternative first exon usage that were regulated by acute and chronic hypoxia where intron retention was the most dominant type of hypoxia-induced alternative splicing. Many of these genes are involved in cellular metabolism, transcriptional regulation, actin cytoskeleton organisation, cancer cell proliferation, migration and invasion, suggesting they may modulate or be involved in additional features of tumorigenic development that extend beyond the known functions of canonical full-length transcripts.
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Empalme Alternativo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Hipoxia/genética , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia Celular/genética , Exones , Femenino , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Humanos , Hipoxia/metabolismo , Intrones , Proteínas de la Membrana/metabolismo , Factor 1 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/metabolismo , Procesamiento Postranscripcional del ARN , Factores de Transcripción/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.
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Regulación de la Expresión Génica/fisiología , Factor de Transcripción HES-1/genética , Transcripción Genética/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Receptores Notch/metabolismo , Factor de Transcripción HES-1/metabolismoRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.
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Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones SCID , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal , Factores de Transcripción/genética , Activación Transcripcional , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hypoxia causes dramatic changes in the expression profiles of genes that encode glycolytic enzymes, vascular endothelial growth factors, erythropoietin, and other factors in a tissue-specific manner through activating hypoxia-inducible transcription factors (HIFs) such as HIF1α and HIF2α. It has been elucidated that the activity of HIFs is fundamentally regulated by their protein stability in an oxygen-dependent manner. However, little is known about how stabilized HIFs regulate transcription of their target genes in hypoxic cells. Additionally, the roles of HIF3α, the third member of the HIFs, are still enigma due to its various splicing variants and the complicated phenotypes of Hif3a-gene modified mouse lines. Here, we summarize how molecular systems fine-tune hypoxia-inducible transcription with the cooperation of HIFs and their negative regulators, including IPAS, one of the HIF3α splicing variants. Since epigenetic mechanisms contribute to stress-inducible and cell-type specific gene regulation, the HIF-dependent reorganization of nucleosome structures in hypoxia-inducible gene promoters is also discussed.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/fisiología , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Animales , Humanos , Factores de Transcripción/metabolismoRESUMEN
The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins.
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Cuerpo Carotídeo/metabolismo , Hipoxia/metabolismo , MicroARNs/genética , Oxígeno/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Hipoxia de la Célula , Células Cultivadas , Humanos , Hipoxia/genética , Masculino , MicroARNs/metabolismo , Persona de Mediana EdadRESUMEN
Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival.
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R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Glutaratos/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , ADN/química , ADN/metabolismo , Metilación de ADN/efectos de los fármacos , Dioxigenasas/metabolismo , Glutaratos/inmunología , Glutaratos/metabolismo , Histonas/metabolismo , Homeostasis/efectos de los fármacos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Activación de Linfocitos , Lisina/metabolismo , Ratones , Oxígeno/metabolismo , Estabilidad Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
UNLABELLED: : Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. SIGNIFICANCE: This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development.
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Técnicas de Cultivo de Célula/métodos , Músculo Esquelético/citología , Distrofia Muscular Facioescapulohumeral , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
Hypoxia-inducible factor-2α (HIF-2α, or EPAS1) is important for cancer progression, and is a putative biomarker for poor prognosis for non-small cell lung cancer (NSCLC). However, molecular mechanisms underlying the EPAS1 overexpression are not still fully understood. We explored a role of a single nucleotide polymorphism (SNP), rs13419896 located within intron 1 of the EPAS1 gene in regulation of its expression. Bioinformatic analyses suggested that a region including the rs13419896 SNP plays a role in regulation of the EPAS1 gene expression and the SNP alters the binding activity of transcription factors. In vitro analyses demonstrated that a fragment containing the SNP locus function as a regulatory region and that a fragment with A allele showed higher transactivation activity than one with G, especially in the presence of overexpressed c-Fos or c-Jun. Moreover, NSCLC patients with the A allele showed poorer prognosis than those with G at the SNP even after adjustment with various variables. In conclusion, the genetic polymorphism of the EPAS1 gene may lead to variation of its gene expression levels to drive progression of the cancer and serve as a prognostic marker for NSCLC.