RESUMO
The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, we have shown that the chromodomain protein Chriz and the zinc-finger protein Z4 are essentially required for the maintenance of polytene chromosome structure. Here we show that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and our data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Histonas/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Cromossomos Politênicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas do Olho/metabolismo , Interfase , FosforilaçãoRESUMO
The subdivision of polytene chromosomes into bands and interbands suggests a structural chromatin organization that is related to the formation of functional domains of gene expression. We made use of the antibody Z4 to gain insight into this level of chromosomal structure, as the Z4 antibody mirrors this patterning by binding to an antigen that is present in most interbands. The Z4 gene encodes a protein with seven zinc fingers, it is essential for fly development and acts in a dose-dependent manner on the development of several tissues. Z4 mutants have a dose-sensitive effect on w(m4) position effect variegation with a haplo-suppressor and triplo-enhancer phenotype, suggesting Z4 to be involved in chromatin compaction. This assumption is further supported by the phenotype of Z4 mutant chromosomes, which show a loss of the band/interband pattern and are subject to an overall decompaction of chromosomal material. By co-immunoprecipitations we identified a novel chromo domain protein, which we named Chriz (Chromo domain protein interacting with Z4) as an interaction partner of Z4. Chriz localizes to interbands in a pattern that is identical to the Z4 pattern. These findings together with the result that Z4 binds directly to DNA in vitro strongly suggest that Z4 in conjunction with Chriz is intimately involved in the higher-order structuring of chromosomes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sequência de Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases/genética , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Aberrações Cromossômicas , Cromossomos/genética , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/citologia , Anormalidades do Olho/genética , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Dados de Sequência Molecular , Mutação/genética , Dedos de Zinco/genéticaRESUMO
We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.