RESUMO
Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w(m4h) allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics.
Assuntos
Cromatina/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas do Olho/metabolismo , Elementos Isolantes , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Cromatina/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Imunoprecipitação , Elementos Isolantes/genética , Elementos Isolantes/fisiologia , Mitose , Modelos Genéticos , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , TransgenesRESUMO
Chromatin domain insulators are thought to insulate adjacent genes, including their regulatory elements, from each other by organizing chromatin into functionally independent domains. Thus insulators should play a global role in gene regulation by keeping regulatory domains separated. However, this has never been demonstrated. We previously designed and characterized a transgene that is under GAL4 UAS control and encodes a dominant-negative form of the Boundary Element-Associated Factors BEAF-32A and BEAF-32B. The BID transgene encodes the BEAF self-interaction domain but lacks a DNA binding domain. Expression of BID in eye imaginal discs leads to a rough eye phenotype. Here we screen for dominant mutations that modify this eye phenotype. This assay provides evidence for cross-talk between different classes of insulators, and for a broad role of the BEAF proteins in maintaining patterns of gene expression during eye development. Most identified genes encode other insulator binding proteins, transcription factors involved in head development, or general transcription factors. Because it is unlikely that insulator function is limited to eye development, the present results support the hypothesis that insulators play a widespread role in maintaining global transcription programs.