RESUMO
The endocycle constitutes an effective strategy for cell growth during development. In contrast to the mitotic cycle, it consists of multiple S-phases with no intervening mitosis and lacks a checkpoint ensuring the replication of the entire genome. Here, we report an essential requirement of chromatin assembly factor-1 (CAF-1) for Drosophila larval endocycles. This complex promotes histone H3-H4 deposition onto newly synthesised DNA in vitro. In metazoans, the depletion of its large subunit leads to the rapid accumulation of cells in S-phase. However, whether this slower S-phase progression results from the activation of cell cycle checkpoints or whether it reflects a more direct requirement of CAF-1 for efficient replication in vivo is still debated. Here, we show that, strikingly, Drosophila larval endocycling cells depleted for the CAF-1 large subunit exhibit normal dynamics of progression through endocycles, although accumulating defects, such as perturbation of nucleosomal organisation, reduction of the replication efficiency of euchromatic DNA and accumulation of DNA damage. Given that the endocycle lacks a checkpoint ensuring the replication of the entire genome, the biological context of Drosophila larval development offered a unique opportunity to highlight the requirement of CAF-1 for chromatin organisation and efficient replication processes in vivo, independently of checkpoint activation.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/fisiologia , Proteínas de Drosophila/metabolismo , Eucromatina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , DNA/genética , Dano ao DNA , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Feminino , Genes de Insetos , Genoma de Inseto , Larva/citologia , Larva/metabolismo , Masculino , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Subunidades Proteicas , Proteína 4 de Ligação ao Retinoblastoma , Fase S , Glândulas Salivares/citologia , Glândulas Salivares/metabolismoRESUMO
The tantalus (tan) gene encodes a protein that interacts specifically with the Polycomb/trithorax group protein Additional sex combs (ASX). Both loss-of-function and gain-of-function mutations in tan cause tissue-specific defects in the eyes, wing veins and bristles of adult flies. As these defects are also typical for components of the Notch (N) signalling pathway, we wished to determine if TAN interacts with this pathway. Through careful examination of ectopic tan phenotypes, we find that TAN specifically disrupts all three major processes associated with the N signalling pathway (boundary formation, lateral inhibition, and lineage decisions). Furthermore, ectopic tan expression abolishes expression of two N target genes, wingless (wg) and cut, at the dorsal-ventral boundary of the wing. An interaction between tan and N was also observed using a genetic assay that previously detected interactions between tan and Asx. The previously observed ability of TAN to move between the cytoplasm and nucleus, and to associate with DNA, provides a potential mechanism for TAN to respond to N signalling.