RESUMEN
BACKGROUND: Members of the NET subfamily of zinc-finger proteins are related to the Sp-family of transcription factors and are required during embryogenesis. In particular, Nlz1/Znf703 and Nlz2/Znf503 are required for formation of rhombomere 4 of the vertebrate hindbrain. While NET family proteins have been hypothesized to regulate transcription, it remains unclear if they function as activators or repressors of transcription. RESULTS: Here we demonstrate that Nlz proteins repress transcription both in cell lines and in developing zebrafish embryos. We first use standard cell culture-based reporter assays to demonstrate that Nlz1/Znf703 represses transcription of a luciferase reporter in four different cell lines. Structure-function analyses and pharmacological inhibition further reveal that Nlz1-mediated repression requires histone deacetylase activity. We next generate a stable transgenic zebrafish reporter line to demonstrate that Nlz1 promotes histone deacetylation at the transgenic promoter and repression of transgene expression during embryogenesis. Lastly, taking a genetic approach we find that endogenous Nlz proteins are required for formation of hindbrain rhombomere 4 during zebrafish embryogenesis by repressing expression of non-rhombomere 4 genes. CONCLUSION: We conclude that Nlz1/Znf703 acts as a repressor of transcription and hypothesize that other NET family members function in a similar manner.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Células HeLa , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Ratones , Células 3T3 NIH , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Transfección , Transgenes , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Dedos de Zinc/genéticaRESUMEN
Increased expression of the histone deacetylase sir2 has been reported to extend the life span of diverse organisms including yeast, Caenorhabditis elegans, and Drosophila melanogaster. A small molecule activator of Sir2, resveratrol, has also been suggested to extend the fitness and survival of these simple model organisms as well as mice fed high calorie diets. However, other studies in yeast have shown that Sir2 itself may prevent life extension, and high expression levels of Sir2 can be toxic to yeast and mouse cells. This conflicting evidence highlights the importance of understanding the mechanisms by which Sir2 expression or activation affects survival of organisms. To investigate the downstream signaling pathways affected by Sir2 in Drosophila, we generated transgenic flies expressing sir2. Here, we show that overexpression of sir2 in Drosophila promotes caspase-dependent but p53-independent apoptosis that is mediated by the JNK and FOXO signaling pathways. Furthermore, we find that a loss-of-function sir2 mutant partially prevents apoptosis induced by UV irradiation in the eye. Together, these results suggest that Sir2 normally participates in the regulation of cell survival and death in Drosophila.
Asunto(s)
Apoptosis , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Histona Desacetilasas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Sirtuinas/metabolismo , Animales , Muerte Celular , Supervivencia Celular , Drosophila/embriología , Factores de Transcripción Forkhead/metabolismo , Inmunohistoquímica , Fenotipo , Rayos UltravioletaRESUMEN
In Friedreich's ataxia, reduction of the mitochondria protein frataxin results in the accumulation of iron and reactive oxygen species, which leads to oxidative damage, neurodegeneration and a diminished lifespan. Recent studies propose that frataxin might play a role in the antioxidative process. Here we show that overexpression of Drosophila frataxin in the mitochondria of female transgenic animals increases antioxidant capability, resistance to oxidative stress insults, and longevity. This suggests that Drosophila frataxin may function to protect the mitochondria from oxidative stresses and the ensuing cellular damage.
Asunto(s)
Drosophila melanogaster/metabolismo , Expresión Génica , Proteínas de Unión a Hierro/genética , Longevidad , Mitocondrias/metabolismo , Estrés Oxidativo , Animales , Animales Modificados Genéticamente , Antioxidantes , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Proteínas de Unión a Hierro/metabolismo , ARN Mensajero , FrataxinaRESUMEN
C2H2 zinc finger proteins make up one of the largest protein families in eukaryotic organisms. Recent study in several different systems has identified a set of novel zinc finger proteins that appear to form a distinct subfamily that we have named the NET family. Members of the NET family (Noc, Nlz, Elbow, and Tlp-1) share two protein motifs--a buttonhead box and an Sp motif--with zinc finger proteins from the Sp family. However, the NET family is uniquely characterized by a single atypical C2H2 zinc finger, in contrast to the Sp family that contains three tandem C2H2 fingers. Here, we review current information about the biochemical function and in vivo role for members of this subfamily. In general, NET family proteins are required during embryonic development. They appear to act by regulating transcription, most likely as repressors, although they are unlikely to bind DNA directly. In the future, it will be important to directly test if NET family proteins control transcription of specific target genes, perhaps via interactions with DNA-binding transcription factors, as well as to further explore their function in vivo.
Asunto(s)
Embrión de Mamíferos/fisiología , Embrión no Mamífero , Morfogénesis , Dedos de Zinc/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Familia de Multigenes , Proteínas Nucleares/clasificación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Filogenia , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In this study, we first cloned nlz2, a second zebrafish member of the nlz-related zinc-finger gene family. nlz2 was expressed together with nlz1 in a broad posterior domain during gastrula stages as well as at the midbrain-hindbrain boundary and in the hindbrain caudal to rhombomere 4 during segmentation. nlz2 was also expressed in regions distinct from nlz1, notably in the forebrain, midbrain, and trunk. Misexpression of nlz2 in zebrafish embryos disrupted gene expression in the rostral hindbrain, similar to the effect of misexpressing nlz1. We next compared the nlz1 and nlz2 sequences to identify and characterize domains conserved within this family. We found a C-terminal domain required for nuclear localization and two conserved domains (the Sp motif and a putative C(2)H(2) zinc finger) required for nlz1 function. We also demonstrate that Nlz1 self-associated via its C terminus, interacted with Nlz2, and bound to histone deacetylases. Last, we found two forms of Nlz1 generated from alternative translation initiation sites in vivo. These forms have distinct activities, apparently depending on the function of the N-terminal Sp motif. Our data demonstrate that nlz2 functions similarly to nlz1 and define conserved domains essential for nuclear localization, self-association, and corepressor binding in this novel family of zinc-finger genes.
Asunto(s)
Proteínas/genética , Secuencia de Bases , Western Blotting , Clonación Molecular , Cartilla de ADN , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Dedos de ZincRESUMEN
The zebrafish nlz gene has a rostral expression limit at the presumptive rhombomere (r) 3/r4 boundary during gastrula stages, and its expression progressively expands rostrally to encompass both r3 and r2 by segmentation stages, suggesting a role for nlz in hindbrain development. We find that Nlz is a nuclear protein that associates with the corepressor Groucho, suggesting that Nlz acts to repress transcription. Consistent with a role as a repressor, misexpression of nlz causes a loss of gene expression in the rostral hindbrain, likely due to ectopic nlz acting prematurely in this domain, and this repression is accompanied by a partial expansion in the expression domains of r4-specific genes. To interfere with endogenous nlz function, we generated a form of nlz that lacks the Groucho binding site and demonstrate that this construct has a dominant negative effect. We find that interfering with endogenous Nlz function promotes the expansion of r5 and, to a lesser extent, r3 gene expression into r4, leading to a reduction in the size of r4. We conclude that Nlz is a transcriptional repressor that controls segmental gene expression in the hindbrain. Lastly, we identify additional nlz-related genes, suggesting that Nlz belongs to a family of zinc-finger proteins.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Represoras/metabolismo , Rombencéfalo/embriología , Pez Cebra/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Filogenia , Proteínas Recombinantes de Fusión , Rombencéfalo/metabolismo , Pez Cebra/metabolismo , Dedos de Zinc/fisiologíaRESUMEN
We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.
Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Regulación del Desarrollo de la Expresión Génica , Rombencéfalo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.