RESUMEN
MARCH5 is a RING finger E3 ligase involved in mitochondrial integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function of MARCH5 during development, we assessed transcript expression in zebrafish embryos. We found that march5 transcripts were of maternal origin and evenly distributed at the 1-cell stage, except for the mid-blastula transition, with expression predominantly in the developing central nervous system at later stages of embryogenesis. Overexpression of march5 impaired convergent extension movement during gastrulation, resulting in reduced patterning along the dorsoventral axis and alterations in the ventral cell types. Overexpression and knockdown of march5 disrupted the organization of the developing telencephalon and diencephalon. Lastly, we found that the transcription of march5 was tightly regulated by the transcriptional regulators CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results demonstrate the essential role of March5 in the development of zebrafish embryos.
Asunto(s)
Sistema Nervioso Central/fisiología , Diencéfalo/embriología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Telencéfalo/embriología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Desarrollo Embrionario , Técnicas de Silenciamiento del Gen , Células HEK293 , Homeostasis , Humanos , Proteínas de la Membrana/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteínas de Pez Cebra/genéticaRESUMEN
Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.
Asunto(s)
Relojes Circadianos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutación , Degradación de ARNm Mediada por Codón sin Sentido , Transactivadores/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas CLOCK/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Neuronas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , Transducción de SeñalRESUMEN
Pellino proteins are associated with immune and stress responses through their effects on NF-κB signaling and B-cell development, and through their role as a scaffold in TLR/IL-1R signaling pathways. However, their function during embryonic development is unclear. Here, we report the developmental expression patterns and functions of peli1b, which encodes a zebrafish ortholog of human Pellino1. Maternal peli1b transcripts were present in zebrafish embryos at the 1-cell stage and zygotic transcripts appeared in the shield area at 6â¯hours post fertilization (hpf), particularly in the neural plate of the dorsal region. peli1b transcripts were concentrated in the somites, lens, myogenic cells, lateral plate mesoderm, and presomitic mesoderm at 12â¯hpf, but expression shifted to the telencephalon, diencephalon, hindbrain, and rhombomeres (r1-7) at 24â¯hpf. Distribution of peli1b transcripts was further restricted to the telencephalon, diencephalon, hindbrain, eyes, and pectoral fins at 48â¯hpf. Knock-down of peli1b with a peli1b antisense morpholino resulted in significant developmental defects and a reduction in size of the telencephalon, diencephalon, rhombomeres (r1-7), and spinal cord at 24â¯hpf. When peli1b-knock-down embryos were analyzed for zic3, a marker associated with the central nervous system, we found lower levels of zic3 transcripts in the shield area at 6â¯hpf and in the posterior diencephalon, dorsal neural plate, midbrain, and hindbrain at 14â¯hpf. Finally, the ERK3/4 inhibitor SB203580 also induced a significant reduction in the level of zic3 transcripts in the neural plate at 6â¯hpf and in the posterior diencephalon, dorsal neural plate, midbrain, hindbrain, segmental plate, dorsal spinal cord, and dorsal posterior neural plate at 14â¯hpf. It is thus likely that the association between Peli1b and brain development in zebrafish embryos occurs via ERK3/4 pathways.
Asunto(s)
Tipificación del Cuerpo/fisiología , Encéfalo/embriología , Sistema de Señalización de MAP Quinasas , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Desarrollo Embrionario , Humanos , Mesodermo/metabolismo , Proteínas Nucleares/metabolismo , Alineación de Secuencia , Somitos/metabolismo , Médula Espinal/metabolismo , Transcriptoma , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , L-Serina Deshidratasa/metabolismo , Mutación , Serina/metabolismo , Transducción de Señal , Inanición/metabolismo , Animales , Conducta Animal , Proteínas de Drosophila/genética , Drosophila melanogaster , L-Serina Deshidratasa/genética , Serina/genética , Inanición/genéticaRESUMEN
Post-translational control is a crucial mechanism for circadian timekeeping. Evolutionarily conserved kinases and phosphatases have been implicated in circadian phosphorylation and the degradation of clock-relevant proteins, which sustain high-amplitude rhythms with 24-hr periodicity in animal behaviors and physiology. Here, we report a novel clock function of the heterodimeric Ca2+/calmodulin-dependent phosphatase calcineurin and its regulator sarah (sra) in Drosophila Genomic deletion of the sra locus dampened circadian locomotor activity rhythms in free-running constant dark after entrainment in light-dark cycles. Poor rhythms in sra mutant behaviors were accompanied by lower expression of two oscillating clock proteins, PERIOD (PER) and TIMELESS (TIM), at the post-transcriptional level. RNA interference-mediated sra depletion in circadian pacemaker neurons was sufficient to phenocopy loss-of-function mutation in sra On the other hand, a constitutively active form of the catalytic calcineurin subunit, Pp2B-14DACT, shortened circadian periodicity in locomotor behaviors and phase-advanced PER and TIM rhythms when overexpressed in clock neurons. Heterozygous sra deletion induced behavioral arrhythmicity in Pp2B-14DACT flies, whereas sra overexpression rescued short periods in these animals. Finally, pharmacological inhibition of calcineurin in either wild-type flies or clock-less S2 cells decreased the levels of PER and TIM, likely by facilitating their proteasomal degradation. Taken together, these data suggest that sra negatively regulates calcineurin by cell-autonomously titrating calcineurin-dependent stabilization of PER and TIM proteins, thereby sustaining high-amplitude behavioral rhythms in Drosophila.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Circadianas Period/metabolismo , Animales , Proteínas de Unión al Calcio , Línea Celular , Ritmo Circadiano , Drosophila/genética , Drosophila/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Circadianas Period/genética , Procesamiento Proteico-Postraduccional , ProteolisisRESUMEN
Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with antimalarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-κB-dependent transcription, and p53-dependent transcription. We also showed by quantitative RT-PCR and western blot assays that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.
Asunto(s)
Alcaloides/farmacología , Dexametasona/efectos adversos , Proteínas Musculares/metabolismo , Atrofia Muscular/tratamiento farmacológico , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alcaloides/química , Animales , Línea Celular , Proteína Forkhead Box O3/metabolismo , Células HEK293 , Humanos , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/inducido químicamente , FN-kappa B/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with anti-malarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-kappaB-dependent transcription and p53-dependent transcription. We also showed that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1 by the quantitative RT-PCR and Western blot. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.
RESUMEN
Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.
Asunto(s)
Chaperonas de Histonas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Chaperonas de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Lisina/metabolismo , Mutación , Proteínas Nucleares/genética , Nucleosomas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
We report the biological functions of a zebrafish homologue of RING-finger protein 152 (rnf152) during embryogenesis. rnf152 was initially identified as a brain-enriched E3 ligase involved in early embryogenesis of zebrafish. Expression of rnf152 was ubiquitous in the brain at 24 hpf but restricted to the eyes, midbrain-hindbrain boundary (MHB), and rhombomeres at 48 hpf. Knockdown of rnf152 in zebrafish embryos caused defects in the eyes, MHB, and rhombomeres (r1-7) at 24 hpf. These defects in rnf152-deficient embryos were analyzed by whole-mount in situ hybridization (WISH) using neuroD, deltaD, notch1a, and notch3 probes. NeuroD expression was abolished in the marginal zone, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the eyes at 27 hpf. Furthermore, deltaD and notch1a expression was remarkably reduced in the ONL, INL, subpallium, tectum, cerebellum, and rhombomeres (r1-7) at 24 hpf, whereas notch3 expression was reduced in the tectum, cerebellum, and rhombomeres at 24 hpf. Finally, we confirmed that expression of Notch target genes, her4 and ascl1a, also decreased significantly in these areas at 24 hpf. Thus, we propose that Rnf152 is essential for development of the eyes, midbrain and hindbrain, and that Delta-Notch signaling is involved.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Pez Cebra/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Embrión no Mamífero , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Pez CebraRESUMEN
Kohlschutter-Tönz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. Here we demonstrate that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, we propose that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS.
Asunto(s)
Dopamina/metabolismo , Drosophila/fisiología , Proteínas Nucleares/genética , Transducción de Señal , Sueño/genética , Vigilia/genética , Ácido gamma-Aminobutírico/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Anticonvulsivantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ritmo Circadiano/genética , Femenino , Neuronas GABAérgicas/metabolismo , Mutación con Pérdida de Función , Modelos Biológicos , Mutación , Proteínas Nucleares/metabolismo , Receptores de GABA/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Kohlschutter-Tönz syndrome (KTS) is a rare autosomal-recessive disorder of childhood onset characterized by global developmental delay, spasticity, epilepsy, and amelogenesis imperfecta. Rogdi, an essential protein, is highly conserved across metazoans, and mutations in Rogdi are linked to KTS. However, how certain mutations in Rogdi abolish its physiological functions and cause KTS is not known. In this study, we determined the crystal structure of human Rogdi protein at atomic resolution. Rogdi forms a novel elongated curved structure comprising the α domain, a leucine-zipper-like four-helix bundle, and a characteristic ß-sheet domain. Within the α domain, the N-terminal H1 helix (residues 19-45) pairs with the C-terminal H6 helix (residues 252-287) in an antiparallel manner, indicating that the integrity of the four-helix bundle requires both N- and C-terminal residues. The crystal structure, in conjunction with biochemical data, indicates that the α domain might undergo a conformational change and provide a structural platform for protein-protein interactions. Disruption of the four-helix bundle by mutation results in significant destabilization of the structure. This study provides structural insights into how certain mutations in Rogdi affect its structure and cause KTS, which has important implications for the development of pharmaceutical agents against this debilitating neurological disease.
Asunto(s)
Amelogénesis Imperfecta/genética , Demencia/genética , Epilepsia/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Leucina Zippers , Mutación , Dominios ProteicosRESUMEN
PRUNE2 has been identified as a susceptible gene for Alzheimer's disease and a marker for leiomyosarcomas. Isoforms of Prune2 regulate neuronal cell differentiation and synaptogenesis. Although expression pattern of Prune2 has been reported in the murine brain, its expression patterns and regulation along vertebrate embryogenesis remain to be further investigated. We thus defined the expression profiles and transcriptional regulation of prune2 in zebrafish embryos. prune2 exhibits maternal expression, but is increased in later embryonic stages, and expressed in the telencephalon, epiphysis cluster, nucleus of the tract of the post optic commissure, spinal cord, cerebellum, tegmentum, anterior lateral line ganglion, posterior lateral line ganglion and rhombomeres 2 through 5. Two color WISH with a post-mitotic neuron specific marker, huC defined that prune2 is expressed in the post mitotic neurons. The level of prune2 transcripts is upregulated in Notch signaling homozygous mutant, mib1-/-(mibta52b), indicating that Notch signaling regulates transcription of prune2. Interestingly, in silico analysis of prune2 promoter found retinoic acid (RA) response elements (AGGTCAcaTGACCA) located at -3 to -16 relative to the first exon. It turned out that RA signaling altered the expression pattern of prune2 in the hindbrain. We further propose that Prune2 might be a putative regulator for CNS development in zebrafish embryogenesis.
Asunto(s)
Encéfalo/embriología , Proteínas de Neoplasias/genética , Transducción de Señal , Proteínas de Xenopus/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Encéfalo/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Receptores Notch/metabolismo , Tretinoina/metabolismo , Pez Cebra/metabolismoRESUMEN
Previous studies have revealed that nucleosomes impede elongation of RNA polymerase II (RNAPII). Recent observations suggest a role for ATP-dependent chromatin remodellers in modulating this process, but direct in vivo evidence for this is unknown. Here using fission yeast, we identify Fun30Fft3 as a chromatin remodeller, which localizes at transcribing regions to promote RNAPII transcription. Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. Mutants, resulting in reduced nucleosome-barrier, such as deletion mutants of histones H3/H4 themselves and the genes encoding components of histone deacetylase Clr6 complex II suppress the defects in growth and RNAPII occupancy of cells lacking Fun30Fft3. These data suggest that RNAPII utilizes the chromatin remodeller, Fun30Fft3, to overcome the nucleosome barrier to transcription elongation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Regulación Fúngica de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Modelos Genéticos , Nucleosomas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcripción GenéticaRESUMEN
Asian dust or yellow sand events in East Asia are a major issue of environmental contamination and human health, causing increasing concern. A high amount of dust particles, especially called as particulate matter 10 (PM10), is transported by the wind from the arid and semi-arid tracks to the Korean peninsula, bringing a bacterial population that alters the terrestrial and atmospheric microbial communities. In this study, we aimed to explore the bacterial populations of Asian dust samples collected during November-December 2014. The dust samples were collected using the impinger method, and the hypervariable regions of the 16S rRNA gene were amplified using PCR followed by pyrosequencing. Analysis of the sequencing data were performed using Mothur software. The data showed that the number of operational taxonomic units and diversity index during Asian dust events were higher than those during non-Asian dust events. At the phylum level, the proportions of Proteobacteria, Actinobacteria, and Firmicutes were different between Asian dust and non-Asian dust samples. At the genus level, the proportions of the genus Bacillus (6.9%), Arthrobacter (3.6%), Blastocatella (2%), Planomicrobium (1.4%) were increased during Asian dust compared to those in non-Asian dust samples. This study showed that the significant relationship between bacterial populations of Asian dust samples and non-Asian dust samples in Korea, which could significantly affect the microbial population in the environment.
Asunto(s)
Microbiología del Aire , Contaminantes Atmosféricos/análisis , Polvo/análisis , Material Particulado/análisis , Monitoreo del Ambiente/métodos , Humanos , Metagenómica , ARN Ribosómico 16S/genética , SeúlRESUMEN
The ATP-binding cassette (ABC) transporter family is one of the largest gene family in animals, and members of this family are known to be involved in various biological processes due to their ability to transport a wide range of substrates across membranes using ATP cleavage-derived energy. We identified 61 ABC transporters in the genome of the monogonont rotifer Brachionus koreanus, and classified these into eight distinct subfamilies (A-H) by phylogenetic analysis. ABC transporters in the rotifer B. koreanus are comprised of 11 ABCA genes, 19 ABCB genes, 14 ABCC genes, 3 ABCD genes, 1 ABCE gene, 3 ABCF genes, 8 ABCG genes, and 2 ABCH genes. Extensive gene duplication and loss events in synteny were observed in several subfamilies. In particular, massive gene duplications of P-glycoproteins (P-gps), multidrug resistance proteins (MRPs), and Bk-Abcg-like proteins were observed. The ability of these B. koreanus proteins to function as multixenobiotic resistance (MXR) ABC transporters was validated using specific fluorescence substrates/inhibitors. The ABC transporter superfamily members identified in this study will be useful in future toxicological studies, and will facilitate comparative studies of the evolution of the ABC transporter superfamily in invertebrates.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Perfilación de la Expresión Génica , Genoma/genética , Rotíferos/genética , Xenobióticos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Biología Computacional , Filogenia , Rotíferos/efectos de los fármacosRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.
RESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Like other herpesviruses, KSHV has two distinct life cycles: latent and lytic. Among KSHV latent genes, viral interferon regulatory factor 3 (vIRF3), which shares homology with cellular IRFs, is a multifunctional protein. To identify unknown functions of vIRF3, we performed luciferase-reporter assays in the presence of vIRF3. These analyses revealed that overexpression of vIRF3 inhibited T-cell factor (TCF)-dependent transcriptional activity. This TCF-dependent transcription was associated with the Wnt signaling pathway, which normally regulates embryonic development, but contributes to oncogenesis under dysregulated conditions. Using a mutagenesis analysis, we identified a CREB-binding protein-interaction motif (LXXLL) in vIRF3 as an important region for its inhibitory activity. Collectively, our findings provide insight into the dysregulation of host signaling pathways in KSHV-infected cells.
Asunto(s)
Proteína de Unión a CREB/química , Herpesvirus Humano 8/fisiología , Interacciones Huésped-Patógeno/genética , Factores Reguladores del Interferón/metabolismo , Factores de Transcripción TCF/antagonistas & inhibidores , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Núcleo Celular/metabolismo , Células HEK293 , Herpesvirus Humano 8/metabolismo , Humanos , Factores Reguladores del Interferón/química , Factores Reguladores del Interferón/genética , Mutación , Factores de Transcripción TCF/metabolismo , Transcripción Genética , Proteínas Virales/química , Proteínas Virales/genética , Latencia del Virus , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. Here, we demonstrate a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, our data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. We thus propose that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species.
Asunto(s)
Ritmo Circadiano/fisiología , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/fisiología , Luz , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutación , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Factores de Transcripción/genéticaRESUMEN
2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure.
Asunto(s)
Copépodos/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ecdisteroides/metabolismo , Éteres Difenilos Halogenados/toxicidad , Metamorfosis Biológica/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Contaminantes Químicos del Agua/toxicidad , Animales , Copépodos/fisiología , Femenino , Marcadores Genéticos , Masculino , Metamorfosis Biológica/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Pruebas de ToxicidadRESUMEN
Mammalian T-type Ca(2+) channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). These channels are reported to be sleep stabilizers important in the generation of the delta rhythms of deep sleep, but controversy remains. The identification of precise physiological functions for the T-type channels has been hindered, at least in part, by the potential for compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene, but its functions are even less well-studied. We cloned Ca-α1T, the only Cav3 channel gene in Drosophila melanogaster, expressed it in Xenopus oocytes and HEK-293 cells, and confirmed it passes typical T-type currents. Voltage-clamp analysis revealed the biophysical properties of Ca-α1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. We found Ca-α1T is broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-α1T show an abnormal increase in sleep duration most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, our study suggests invertebrate T-type Ca(2+) channels promote wakefulness rather than stabilizing sleep.