RESUMEN
Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.
Asunto(s)
Arabidopsis/fisiología , Relojes Biológicos/genética , Ritmo Circadiano , Regulación de la Expresión Génica de las Plantas , Transcripción Genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Metabolismo de los Hidratos de Carbono , Perfilación de la Expresión Génica , Genes de Plantas , Luz , Nitrógeno/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fotosíntesis/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Azufre/metabolismoRESUMEN
PAS (PER, ARNT, SIM) proteins play important roles in adaptation to low atmospheric and cellular oxygen levels, exposure to certain environmental pollutants, and diurnal oscillations in light and temperature. In an attempt to better understand how organisms sense environmental changes, we have characterized a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23. This protein displays significant homology to the Drosophila circadian factor CYCLE and its putative mammalian ortholog MOP3/bMAL1. Like its homologs, MOP9 forms a transcriptionally active heterodimer with the circadian CLOCK protein, the structurally related MOP4, and hypoxia-inducible factors, such as HIF1alpha. In a manner consistent with its role as a biologically relevant partner of these proteins, MOP9 is coexpressed in regions of the brain such as the thalamus, hypothalamus, and amygdala. Importantly, MOP9 is coexpressed with CLOCK in the suprachiasmatic nucleus, the site of the master circadian oscillator in mammals.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Drosophila , Secuencias Hélice-Asa-Hélice , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción ARNTL , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Northern Blotting , Proteínas CLOCK , Hipoxia de la Célula/fisiología , Línea Celular , Mapeo Cromosómico , Ritmo Circadiano/fisiología , Proteínas de Unión al ADN/metabolismo , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Homología de Secuencia de Aminoácido , Núcleo Supraquiasmático/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Cellular, local, and organismal responses to low O2 availability occur during processes such as anaerobic metabolism and wound healing and pathological conditions such as stroke and cancer. These responses include increases in glycolytic activity, vascularization, breathing, and red blood cell production. These responses are mediated in part by the hypoxia-inducible factors (HIFs), which receive information on O2 levels from a group of iron- and O2-dependent hydroxylases. Hypoxia mimics, such as cobalt chloride, nickel chloride, and deferoxamine, act to simulate hypoxia by altering the iron status of these hydroxylases. To determine whether these mimics are appropriate substitutes for the lower O2 tension evoked naturally, we compared transcriptional responses of a Hep3B cell line using high-density oligonucleotide arrays. A battery of core genes was identified that was shared by all four treatments (hypoxia, cobalt, nickel, and deferoxamine) including glycolytic enzymes, cell cycle regulators, and apoptotic genes. Importantly, cobalt, nickel, and deferoxamine influenced transcription of distinct sets of genes that were not affected by cellular hypoxia. These global responses to hypoxia indicate a balancing act between adaptation and programmed cell death and suggest caution in the use of hypoxia mimics as substitutes for the low O2 tension that occurs in vivo.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hipoxia , Neoplasias Hepáticas/metabolismo , Algoritmos , Línea Celular Tumoral , Cobalto/química , Cartilla de ADN/química , Deferoxamina/química , Regulación hacia Abajo , Eritrocitos/metabolismo , Humanos , Níquel/química , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Oxígeno/metabolismo , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.
Asunto(s)
Cromosomas Humanos Par 9 , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Clonación Molecular , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Bases de Datos Factuales , Elementos de Facilitación Genéticos , Secuencias Hélice-Asa-Hélice , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras , Transfección , Regulación hacia ArribaRESUMEN
Hypoxia inducible factor 1 alpha (HIF-1 alpha) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that mediates certain cellular responses to low oxygen tension, iron chelators, Co2+, Ni2+, Mg2+, and low intracellular glucose concentration. Upon exposure to the above conditions, HIF-1 alpha is upregulated and heterodimerizes with the Ah receptor nuclear translocator (ARNT, also known as HIF-1 beta), the heterodimeric complex binds TACGTG-containing genomic enhancer elements, and activates transcription of target genes. As a first step in developing genetic models to study the biology related to cellular hypoxia, we have cloned the murine HIF-1 alpha cDNA, determined the tissue-specific expression of its mRNA, functionally analyzed its protein product, and characterized its promoter and its genomic structure. A comparison between the murine and human HIF-1 alpha protein sequence reveals 95%, 99%, and 83% identity in the bHLH, PAS, and variable domains, respectively. RNAse protection assays demonstrate that in adult mice, the mHIF-1 alpha mRNA is expressed at high levels in kidney, heart, brain, thymus, and placenta, with moderate expression in liver, spleen, testis, and lung and much lower expression in skeletal muscle testis. Northern blot analysis indicates that the mRNA of the murine HIF-1 alpha is transcribed in two forms, a major 4-kb species and a minor 5-kb species; both are present in all tissues examined. The Hif-1 alpha promoter is GC rich, does not have a TATA element near its transcriptional start site, and does not respond to hypoxia or Co2+. The mHIF-1 alpha structural gene is composed of 15 exons. The splice junction sites within the bHLH and the PAS domains of HIF-1 alpha gene are highly conserved with respect to a number of previously characterized members of the bHLH-PAS superfamily. However, unlike other bHLH-PAS genes, where the variable domain is encoded by 2 exons, the variable region of the mHIF-1 alpha gene is encoded by 7 exons. Furthermore, most of these splice junction sites in the variable region are conserved with that of HIF-2 alpha, a recently cloned hypoxia-responsive bHLH-PAS protein (also known as MOP2, EPAS1, and HLF). These data suggest that HIF-1 alpha, along with HIF-2 alpha, represents a new subclass of the bHLH-PAS superfamily.
Asunto(s)
Proteínas de Unión al ADN/genética , Secuencias Hélice-Asa-Hélice/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Empalme del ARN , ARN Mensajero , Distribución Tisular , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
In both the suprachiasmatic nucleus (SCN) and peripheral tissues, the circadian oscillator drives rhythmic transcription of downstream target genes. Recently, a number of studies have used DNA microarrays to systematically identify oscillating transcripts in plants, fruit flies, rats, and mice. These studies have identified several dozen to many hundred rhythmically expressed genes by sampling tissues every 4 hours for 1, 2, or more days. To extend this work, we have performed DNA microarray analysis on RNA derived from the mouse pituitary sampled every hour for 2 days. COSOPT and Fisher's G-test were used at a false-discovery rate of less than 5% to identify more than 250 genes in the pituitary that oscillate with a 24-hour period length. We found that increasing the frequency of sampling across the circadian day dramatically increased the statistical power of both COSOPT and Fisher's G-test, resulting in considerably more high-confidence identifications of rhythmic transcripts than previously described. Finally, to extend the utility of these data sets, a Web-based resource has been constructed (at http://wasabi.itmat.upenn.edu/circa/mouse ) that is freely available to the research community.
Asunto(s)
Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Hipófisis/fisiología , Animales , Biología Computacional , Bases de Datos Genéticas , Expresión Génica , Perfilación de la Expresión Génica/estadística & datos numéricos , Internet , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Factores de TiempoRESUMEN
Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Interferencia de ARN , ARN no Traducido/fisiología , Factores de Transcripción/antagonistas & inhibidores , Animales , Línea Celular , Humanos , Ratones , Factores de Transcripción NFATC , ARN Largo no Codificante , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/genética , beta Carioferinas/metabolismoRESUMEN
The Ah-receptor (AHR) is a ligand activated transcription factor that mediates the biological effects of agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Upon binding agonists, the AHR dimerizes with a structurally related protein known as ARNT and this heterodimer then binds cognate enhancer elements and activates the expression of target genes. In this report we describe the cloning of the rat AHR cDNA and a fragment of the rat ARNT cDNA for use as probes in ribonuclease protection analysis. Ribonuclease protection analysis indicated that the rat AHR mRNA is expressed at the highest levels in the lung > thymus > kidney > liver while lower levels were expressed in heart and spleen. The rat AHR and ARNT mRNAs were expressed in a largely coordinate manner across the eight tissues examined with the exception of the placenta where AHR levels were relatively low compared to ARNT. In these experiments, a rare splice variant of the AHR was cloned that encoded a protein with a deletion in the ligand binding domain. In vitro expression studies demonstrated that in contrast to the full length AHR, the splice variant did not bind ligand nor did it bind to a cognate enhancer element in the presence of ARNT.
Asunto(s)
Proteínas de Unión al ADN , Expresión Génica , Proteínas/genética , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Secuencia de Bases , Clonación Molecular , Sondas de ADN , Elementos de Facilitación Genéticos , Femenino , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas/química , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Receptores de Hidrocarburo de Aril/química , Mapeo Restrictivo , Ribonucleasas/metabolismo , Distribución TisularRESUMEN
Over the past decade, PAS domains have been identified in dozens of signal transduction molecules and various forms have been found in animals, plants, and prokaryotes. In this review, we summarize this rapidly expanding research area by providing a detailed description of three signal transduction pathways that utilize PAS protein heterodimers to drive their transcriptional output. It is hoped that these model pathways can provide a framework for use in understanding the biology of the less well-understood members of this emerging superfamily, as well as of those to be characterized in the days to come. We use this review to develop the idea that most eukaryotic PAS proteins can be classified by functional similarities, as well as by predicted phylogenetic relationships. We focus on the alpha-class proteins, which often act as sensors of environmental signals, and the beta-class proteins, which typically act as broad-spectrum partners that target these heterodimers to their genomic targets.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Transducción de Señal , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Hipoxia de la Célula , Ritmo Circadiano , Proteínas de Unión al ADN/química , Proteínas de Drosophila , Ambiente , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Circadianas Period , Receptores de Hidrocarburo de Aril/fisiología , Factores de Transcripción/químicaRESUMEN
We report that MOP3 is a general dimerization partner for a subset of the basic-helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) superfamily of transcriptional regulators. We demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1alpha (HIF1alpha), and HIF2alpha. A DNA selection protocol revealed that the MOP3-MOP4 heterodimer bound a CACGTGA-containing DNA element. Transient transfection experiments demonstrated that the MOP3-MOP4 and MOP3-CLOCK complexes bound this element in COS-1 cells and drove transcription from a linked luciferase reporter gene. We also deduced the high-affinity DNA binding sites for MOP3-HIF1alpha complex (TACGTGA) and used transient transfection experiments to demonstrate that the MOP3-HIF1alpha and MOP3-HIF2alpha heterodimers bound this element, drove transcription, and responded to cellular hypoxia. Finally, we found that MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo.
Asunto(s)
Ritmo Circadiano , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas CLOCK , Células COS , ADN/metabolismo , Dimerización , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Unión Proteica , TransfecciónRESUMEN
In an effort to better understand the mechanism of toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new "members of the PAS superfamily," or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Nucleares/metabolismo , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Translocador Nuclear del Receptor de Aril Hidrocarburo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Clonación Molecular , ADN Complementario/química , Proteínas de Drosophila , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN Mensajero/metabolismo , Alineación de Secuencia , Programas InformáticosRESUMEN
Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.
Asunto(s)
Ritmo Circadiano/fisiología , Proteínas de Unión al ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Conducta Animal , Proteínas de Ciclo Celular , Sondas de ADN , Expresión Génica/fisiología , Mamíferos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Actividad Motora , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fenotipo , Núcleo Supraquiasmático/química , Núcleo Supraquiasmático/fisiologíaRESUMEN
Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.