RESUMEN
Aberrant chromatin remodeling is involved in the pathogenesis of Huntington's disease (HD) but the mechanism is not known. Herein, we report that mutant huntingtin (mtHtt) induces the transcription of alpha thalassemia/mental retardation X linked (ATRX), an ATPase/helicase and SWI/SNF-like chromatin remodeling protein via Cdx-2 activation. ATRX expression was elevated in both a cell line model and transgenic model of HD, and Cdx-2 occupancy of the ATRX promoter was increased in HD. Induction of ATRX expanded the size of promyelocytic leukemia nuclear body (PML-NB) and increased trimethylation of H3K9 (H3K9me3) and condensation of pericentromeric heterochromatin, while knockdown of ATRX decreased PML-NB and H3K9me3 levels. Knockdown of ATRX/dXNP improved the hatch rate of fly embryos expressing mtHtt (Q127). ATRX/dXNP overexpression exacerbated eye degeneration of eye-specific mtHtt (Q127) expressing flies. Our findings suggest that transcriptional alteration of ATRX by mtHtt is involved in pericentromeric heterochromatin condensation and contributes to the pathogenesis of HD.
Asunto(s)
ADN Helicasas/metabolismo , Heterocromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Factor de Transcripción CDX2 , Línea Celular , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Drosophila , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Metilación , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
We have developed a strategy using Drosophila as a model system to identify genes that are crucial for extension of longevity. A collection of transgenic lines with a P-element based gene search (GS) vector containing UAS (Upstream Activating Sequence) was screened for longevity in combination with an hsp70 promoter-driven GAL4 transgene. Misexpression of the vector-flanking sequence was induced throughout the adult stage to assess its effects on the aging process rather than development. We showed that the longevity was greatly affected by GS inserts, and it was positively correlated with paraquat resistance. Of 646 GS inserts, we selected 23 inserts with relatively longer longevity for further molecular analysis. All of the misexpressed sequences matched either known genes or ESTs (Expressed Sequence Tags). Among 13 genes whose functions are already known or suggested, six were related to stress resistance or redox balance (DmGST2, hsp26, nla, and Drosophila homologs of mammalian TRX, GILT and POSH), suggesting the importance of stress resistance for the extension of longevity. This is the first demonstration that a systematic gain-of-function screen could efficiently detect longevity genes.
Asunto(s)
Pruebas Genéticas/métodos , Longevidad/genética , Modelos Genéticos , Animales , Drosophila/genética , Femenino , Masculino , Mutagénesis , Estrés Oxidativo/genéticaRESUMEN
Extended longevity mutants are extremely useful to understand the molecular mechanism of longevity determination. Here we report identification and characterization of the Drosophila Plenty of SH3s (DPOSH) gene, a candidate that might be associated with the extended longevity phenotype. DPOSH encodes a protein containing a RING finger domain and four SH3 domains. We showed that neural-specific overexpression of DPOSH could extend the mean longevity of adult flies by 14% at 25 degrees C without affecting viability or morphology. In contrast, forced expression of DPOSH in developing imaginal discs produced various phenotypes including lethality and morphological defects such as loss of crossvein, notched wing, and disordered hair polarity. Puckered, a target gene of JNK/SAPK pathway, was activated by overexpression of DPOSH and the forced expression phenotypes were suppressed by introducing a mutation of Drosophila JNK (bsk) or JNKK (hep), suggesting that the JNK/SAPK signaling pathway is one of the critical elements in the determination of longevity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Proteínas de Insectos/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Activación Enzimática , Femenino , Expresión Génica , Genes de Insecto , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Longevidad/genética , Longevidad/fisiología , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Transducción de SeñalRESUMEN
We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.