RESUMO
The trithorax group gene brahma (brm) encodes an activator of Drosophila homeotic genes that functions as the ATPase subunit of a large protein complex. To determine if BRM physically interacts with other trithorax group proteins, we purified the BRM complex from Drosophila embryos and analyzed its subunit composition. The BRM complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the BRM complex are not encoded by trithorax group genes. Furthermore, a screen for enhancers of a dominant-negative brm mutation identified only one trithorax group gene, moira (mor), that appears to be essential for brm function in vivo. Four of the subunits of the BRM complex are related to subunits of the yeast chromatin remodeling complexes SWI/SNF and RSC. The BRM complex is even more highly related to the human BRG1 and hBRM complexes, but lacks the subunit heterogeneity characteristic of these complexes. We present biochemical evidence for the existence of two additional complexes containing trithorax group proteins: a 2 MDa ASH1 complex and a 500 kDa ASH2 complex. These findings suggest that BRM plays a role in chromatin remodeling that is distinct from the function of most other trithorax group proteins.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Southern Blotting , Western Blotting , Cruzamentos Genéticos , DNA Helicases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/genética , Etiquetas de Sequências Expressas , Proteínas de Grupo de Alta Mobilidade , Histona-Lisina N-Metiltransferase , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Dados de Sequência Molecular , Proteínas Nucleares/química , Testes de Precipitina , Análise de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Leveduras/genéticaRESUMO
The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas de Insetos/genética , Proteínas Nucleares , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Ligação a DNA/química , Drosophila/embriologia , Proteínas de Drosophila , Proteínas de Insetos/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/química , Fatores de Transcrição/químicaRESUMO
During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.
