Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

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.     

The Arabidopsis SWI2/SNF2 Chromatin Remodeler BRAHMA Regulates Polycomb Function during Vegetative Development and Directly Activates the Flowering Repressor Gene SVP

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.     

A Screen for Genes That Function Downstream of Ras1 during Drosophila Eye Development

Cell-fate specification of the R7 photoreceptor cell is controlled by the sevenless receptor tyrosine kinase (SevRTK) and Ras1, the Drosophila homologue of mammalian H-ras, K-ras and N-ras oncogenes. An activated form of Ras1 expressed under control of the sevenless enhancer/promoter (sev-Ras1(V12)) induces production of supernumerary R7 photoreceptor cells, which causes the eye to become rough in appearance. To isolate mutations in genes functioning downstream of Ras1, we carried out a screen for dominant suppressors and enhancers of this rough eye phenotype. Approximately 850,000 mutagenized flies were screened, and 282 dominant suppressors and 577 dominant enhancers were isolated. Mutations in the Drosophila homologues of Raf, MEK, MAPK, type I Geranylgeranyl Transferase and Protein Phosphatase 2A were isolated, as were mutations in several novel signaling genes. Some of these mutant genes appear to be general signaling factors that function in other Ras1 pathways, while one seems to be more specific for photoreceptor development. At least two suppressors appear to function either between Ras1 and Raf or in parallele to Raf.     

Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signaling through the DR4 receptor by competing for ligand binding

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a "dominant negative" effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.     

Drosophila cyclin E interacts with components of the Brahma complex

Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, we carried out a genetic screen using a hypomorphic mutation of Drosophila cyclin E (DmcycE(JP)), which gives rise to adults with a rough eye phenotype. Amongst the dominant suppressors of DmcycE(JP), we identified brahma (brm) and moira (mor), which encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycE(JP) eye phenotype. We show that Brm complex mutants suppress the DmcycE(JP) phenotype by increasing S phases without affecting DmcycE protein levels and that DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G(1) arrest.     

Dominant Enhancers of Egfr in Drosophila Melanogaster: Genetic Links between the Notch and Egfr Signaling Pathways

The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F(1) screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context.     

Mutations Modulating Raf Signaling in Drosophila Eye Development

The R7 fate is specified during Drosophila eye development by an inductive signal transduced intracellularly via the Raf kinase. We have performed a genetic screen for dominant mutations that alter the efficiency with which cells respond to a constitutively activated Raf kinase. Such mutations may affect genes involved in signal transduction downstream of Raf. We have isolated 44 mutations that define eight genes. One of these encodes a mitogen-activated protein kinase homologue; another is a putative target gene of this signaling pathway. We present the results of this screen in detail, as well as a preliminary genetic analysis of the six loci still to be characterized molecularly.     

The Arabidopsis BRAHMA chromatin-remodeling ATPase is involved in repression of seed maturation genes in leaves

Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue.