ABSTRACT
Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin/ultrastructure , Chromosomes/ultrastructure , DNA-Binding Proteins , Drosophila Proteins , Gene Expression , Transcription Factors/metabolism , X Chromosome/ultrastructure , Acetylation , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Animals , Cell Survival , Drosophila/anatomy & histology , Drosophila/embryology , Drosophila/genetics , Euchromatin , Female , Fluorescent Antibody Technique , Genes, Essential , Heterochromatin/ultrastructure , Homeodomain Proteins/isolation & purification , Homeodomain Proteins/metabolism , Male , Mitosis , Mutation , Phenotype , Transcription Factors/genetics , Transcription Factors/isolation & purificationABSTRACT
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.
Subject(s)
Cell Cycle Proteins , Drosophila Proteins , Drosophila/metabolism , Insect Proteins/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Blotting, Southern , Blotting, Western , Crosses, Genetic , DNA Helicases , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Drosophila/embryology , Drosophila/genetics , Expressed Sequence Tags , High Mobility Group Proteins , Histone-Lysine N-Methyltransferase , Humans , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/isolation & purification , Molecular Sequence Data , Nuclear Proteins/chemistry , Precipitin Tests , Sequence Analysis , Sequence Homology, Amino Acid , Trans-Activators/chemistry , Trans-Activators/genetics , Transcription Factors/chemistry , Yeasts/geneticsABSTRACT
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.
