RESUMEN
A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Argonautas/genética , Sitios de Unión/genética , Western Blotting , Factor de Unión a CCCTC , Línea Celular , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Proteínas de Drosophila/genética , Eucromatina/genética , Eucromatina/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Perfilación de la Expresión Génica , Genoma de los Insectos/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Mutación , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
INTRODUCTION: RASopathies are a group of genetic conditions due to alterations of the Ras/MAPK pathway. Neurocutaneous findings are hallmark features of the RASopathies, but musculoskeletal abnormalities are also frequent. The objective was to evaluate handgrip strength in the RASopathies. METHODS: Individuals with RASopathies (e.g., Noonan syndrome, Costello syndrome, cardio-facio-cutaneous [CFC] syndrome, and neurofibromatosis type 1 [NF1]) and healthy controls were evaluated. Two methods of handgrip strength were tested: GRIP-D Takei Hand Grip Dynamometer and the Martin vigorimeter. A general linear model was fitted to compare average strength among the groups, controlling for confounders such as age, gender, height, and weight. RESULTS: Takei dynamometer: handgrip strength was decreased in each of the syndromes compared with controls. Decreased handgrip strength compared with sibling controls was also seen with the Martin vigorimeter (P < 0.0001). CONCLUSIONS: Handgrip strength is decreased in the RASopathies. The etiology of the reduced muscle force is unknown, but likely multifactorial.
Asunto(s)
Síndrome de Costello/fisiopatología , Displasia Ectodérmica/fisiopatología , Insuficiencia de Crecimiento/fisiopatología , Fuerza de la Mano/fisiología , Cardiopatías Congénitas/fisiopatología , Debilidad Muscular/fisiopatología , Neurofibromatosis 1/fisiopatología , Síndrome de Noonan/fisiopatología , Proteínas ras/genética , Adolescente , Adulto , Niño , Preescolar , Síndrome de Costello/genética , Síndrome de Costello/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Facies , Insuficiencia de Crecimiento/genética , Insuficiencia de Crecimiento/metabolismo , Femenino , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Proteínas ras/metabolismoRESUMEN
Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisina/metabolismo , Metilación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Tretinoina/farmacologíaRESUMEN
ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with beta-catenin and that CHD8 is also recruited specifically to the promoter regions of several beta-catenin-responsive genes. Our results indicate that CHD8 negatively regulates beta-catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several beta-catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of beta-catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure.
Asunto(s)
Adenosina Trifosfato/química , Cromatina/química , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN Helicasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HeLa , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Modelos Biológicos , Interferencia de ARN , Transcripción GenéticaRESUMEN
Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.