The snr1 gene of Drosophila melanogaster encodes a conserved component of the multiprotein Brahma (Brm) complex, a counterpart to the SWI/SNF complexes that participate in ATP-dependent chromatin remodeling. Loss-of-function and null mutations in the snr1 gene reveal its essential role in Drosophila development. We identified new mutant alleles and ectopically expressed deleted forms to dissect the specific functions of SNR1. Somatic and germ cell clone analyses confirmed its requirement in a continuous and widespread fashion for proper cell fate determination and oogenesis. Expression of SNR1 transgenes revealed unexpected roles in wing patterning, abdomen development, oogenesis, and sustained adult viability. A widespread distribution of SNR1 and BRM on the salivary gland polytene chromosomes showed that the Brm complex associated with many genes, but not always at transcribed loci, consistent with genetic data suggesting roles in both gene activation and repression. Despite essential Brm complex functions in leg development, genetic and protein localization studies revealed that snr1 was not required or expressed in all tissues dependent on Brm complex activities. Thus, SNR1 is essential for some, but not all Brm functions, and it likely serves as an optional subunit, directing Brm complex activity to specific gene loci or cellular processes.

The Brahma (Brm) complex of Drosophila melanogaster is a SWI/SNF-related chromatin remodeling complex required to correctly maintain proper states of gene expression through ATP-dependent effects on chromatin structure. The SWI/SNF complexes are comprised of 8-11 stable components, even though the SWI2/SNF2 (BRM, BRG1, hBRM) ATPase subunit alone is partially sufficient to carry out chromatin remodeling in vitro. The remaining subunits are required for stable complex assembly and/or proper promoter targeting in vivo. Our data reveals that SNR1 (SNF5-Related-1), a highly conserved subunit of the Brm complex, is required to restrict complex activity during the development of wing vein and intervein cells, illustrating a functional requirement for SNR1 in modifying whole complex activation functions. Specifically, we found that snr1 and brm exhibited opposite mutant phenotypes in the wing and differential misregulation of genes required for vein and intervein cell development, including rhomboid, decapentaplegic, thick veins, and blistered, suggesting possible regulatory targets for the Brm complex in vivo. Our genetic results suggest a novel mechanism for SWI/SNF-mediated gene repression that relies on the function of a 'core' subunit to block or shield BRM (SWI2/SNF2) activity in specific cells. The SNR1-mediated repression is dependent on cooperation with histone deacetylases (HDAC) and physical associations with NET, a localized vein repressor.

Metazoan SWI/SNF chromatin remodeling complexes exhibit ATP-dependent activation and repression of target genes. The Drosophila Brahma (SWI/SNF) complex subunits BRM and SNR1 are highly conserved with direct counterparts in yeast (SWI2/SNF2 and SNF5) and mammals (BRG1/hBRM and INI1/hSNF5). BRM encodes the catalytic ATPase required for chromatin remodeling and SNR1 is a regulatory subunit. Importantly, SNR1 mediates ATP-independent repression functions of the complex in cooperation with histone deacetylases and direct contacts with gene-specific repressors. SNR1 and INI1, as components of their respective SWI/SNF complexes, are important for developmental growth control and patterning, with direct function as a tumor suppressor. To identify direct regulatory targets of the Brm complex, we performed oligonucleotidebased transcriptome microarray analyses using RNA isolated from mutant fly strains harboring dominant-negative alleles of snr1 and brm. Steady-state RNA isolated from early pupae was examined, as this developmental stage critically requires Brm complex function. We found the hormone-responsive Ecdysone-induced genes (Eig) were strongly misregulated and that the Brm complex is directly associated with the promoter regions of these genes in vivo. Our results reveal that the Brm complex assists in coordinating hormone-dependent transcription regulation of the Eig genes.The metazoan SWI/SNF ATP-dependent chromatin remodeling complexes are large (1.2 MDa, 8 -11 subunits) multimeric assemblies that act to modify nucleosome structure, allowing for activation or repression of gene transcription (1, 2). Prevailing models suggest that the SWI/SNF complexes are recruited to specific in vivo targets through interactions with DNA-binding transcription factors, where the ATP-dependent activities of the complex assist in gene regulation through changes in DNA-histone contacts (3, 4). Subsequently, the affected nucleosomes can be covalently modified to maintain active or repressed transcription through cooperation with histone acetyltransferases and histone deacetylase complexes (5).Whole genome analyses of yeast and mammalian SWI/SNF complex mutants revealed that the expression of ϳ5% of all genes was affected by removal of the complex (6, 7). Importantly, whereas there was a detectable reduction in the expression of a small subset of genes, more transcripts were induced upon loss of the gene encoding the core ATPase (SNF2/SWI2), suggesting that the complex had direct roles in both gene activation and repression. In fact, both epigenetic functions of the yeast complex appear to rely on the ATPase activity of SNF2/ SWI2 (8). Although the yeast SWI/SNF complex is not required for vegetative growth, the metazoan SWI/SNF complex counterparts, including the Brahma (Brm) complex in Drosophila (9, 10) and the related mammalian hBrm/Brg1 complexes (11), are essential (12, 13). This requirement may be due to direct involvement of the Brm complexes in facilitating global gene expression by RNA polymerase II (14).The snr1 gene (S...

SUMMARYMLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N-and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code.