RESUMO
The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein-the Polycomb (PC) protein-with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.
Assuntos
Proteínas de Drosophila , Histonas/metabolismo , Proteínas de Insetos/metabolismo , Nucleossomos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Drosophila , Dados de Sequência Molecular , Complexo Repressor Polycomb 1 , Estrutura Terciária de Proteína , TripsinaRESUMO
Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos/enzimologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Inativação Gênica , Proteínas de Insetos/metabolismo , Nucleoproteínas/metabolismo , Animais , Padronização Corporal/genética , Proteínas de Ciclo Celular/análise , Núcleo Celular/química , Células Cultivadas , Segregação de Cromossomos/genética , DNA Topoisomerases Tipo II/análise , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Drosophila , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Insetos/análise , Proteínas de Insetos/genética , Masculino , Mitose/genética , Mutação , Nucleoproteínas/análise , Nucleoproteínas/genética , Complexo Repressor Polycomb 1 , Testes de PrecipitinaRESUMO
To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, we used formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Here we show that binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and that depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. We further show that PcG proteins interact in vitro with GTFs. We suggest that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription.
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , DNA/metabolismo , Drosophila , Inativação Gênica , Genes de Insetos , Histonas/metabolismo , Dados de Sequência Molecular , Complexo Repressor Polycomb 1 , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The abnormal oocyte (abo) gene of Drosophila melanogaster is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. Here we show that abo encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. We also show a significant increase of histone transcripts in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect by deficiencies of the histone gene cluster. On the basis of these results, we suggest that the Abo protein functions specifically as a negative regulator of histone transcription and propose a molecular model to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect. Our model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila.