RESUMO
ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in Drosophila melanogaster. To identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses.
Assuntos
Adenosina Trifosfatases/metabolismo , Sequência Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolução Molecular , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Animais , Bioensaio , Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Olho/patologia , Fluorometria , Genes Supressores , Testes Genéticos , Mutação/genética , Fenótipo , Ligação Proteica , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Nucleossomos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Animais , Western Blotting , Cromatina/genética , Cromatina/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Imunoprecipitação , Hibridização in Situ Fluorescente/métodos , Fatores de Transcrição/genéticaRESUMO
Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/genética , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Cromossomos/química , Cromossomos/genética , Proteínas de Drosophila/análise , Drosophila melanogaster/metabolismo , Feminino , Histona Desacetilase 1 , Histona Desacetilases/análise , Histonas/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Proteômica , Proteínas Repressoras/análise , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
The developing Drosophila eye-antennal disc is a particularly suited system for the genetic and cellular studies of complex biological processes. Methods to analyze Drosophila eye discs by flow cytometry are mainly based on the dissociation of tissues with trypsin. Dissociation operated by trypsin is very effective, though it causes a lot of stress to live cells often compromising the use of treated cells for further analyses. Here, we report a method to produce dissociated eye-disc cells that retain cell-membrane markers and that can be used for flow cytometry and cytological analysis of mitotic chromosomes. The method described is a great complementing tool for the cellular characterization of phenotypes resulting from classic clonal and miss-expression approaches in the Drosophila eye.