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
While heterochromatic gene silencing in cis is often accompanied by nucleosomal compaction, characteristic histone modifications, and recruitment of heterochromatin proteins, little is known concerning genes silenced by heterochromatin in trans. An insertion of heterochromatic satellite DNA in the euchromatic brown (bw) gene of Drosophila melanogaster results in bwDominant (bwD), which can inactivate loci on the homolog by relocation near the centric heterochromatin (trans-inactivation). Nucleosomal compaction was found to accompany trans-inactivation, but stereotypical heterochromatic histone modifications were mostly absent on silenced reporter genes. HP1 was enriched on trans-inactivated reporter constructs and this enrichment was more pronounced on adult chromatin than on larval chromatin. Interestingly, this HP1 enrichment in trans was unaccompanied by an increase in the 2MeH3K9 mark, which is generally thought to be the docking site for HP1 in heterochromatin. However, a substantial increase in the 2MeH3K9 mark was found on or near the bwD satellite insertion in cis, but did not spread further. These observations suggest that the interaction of HP1 with chromatin in cis is fundamentally different from that in trans. Our molecular data agree well with the differential phenotypic effect on bwD trans-inactivation of various genes known to be involved in histone modification and cis gene silencing.