The domino SWI2/SNF2 Gene Product Represses Cell Death in Drosophila melanogaster.

The Drosophila domino locus encodes DNA-dependent ATPases of the SWI2/SNF2 class. This class of chromatin remodeler is associated with an array of cellular activities encompassing transcription, replication, repair and recombination. Moreover, domino was observed initially to maintain a repressive chromatin state via genetic interaction studies with homeotic genes. Although domino mutations were also characterized with a cell death phenotype, its association with a death pathway has not been investigated. Here we have used targeted RNA interference to depress domino function in the wing. Resultant wing damage phenotypes were found to be enhanced through overexpression of pro-apoptotic loci, and suppressed through loss of function of these loci. Loss of wing margin and blade tissue was correlated with activation of the effector Caspase Dcp-1, a marker for apoptosis. The affected wing regions also exhibited lower levels of the DIAP1 protein, an inhibitor of apoptosis. The lower level of DIAP1 protein was not correlated with an effect on the activity of a DIAP1 gene transgenic reporter (thread-LacZ), suggesting that loss of DIAP1 occurred post transcriptionally. In some cases excessive cell proliferation within the targeted tissue, measured through BrdU incorporation, was also observed. Finally, we used a transgenic reporter construct to monitor the chromatin state upstream of the proapoptotic reaper locus. In genotypes exhibiting targeted domino loss and wing phenotypes, we observed increased reporter activity only in the affected areas. These data support the conclusion that domino normally functions to maintain pro-apoptotic genes in a repressed state.

Drosophila domino Exhibits Genetic Interactions with a Wide Spectrum of Chromatin Protein-Encoding Loci.

The Drosophila domino gene encodes protein of the SWI2/SNF2 family that has widespread roles in transcription, replication, recombination and DNA repair. Here, the potential relationship of Domino protein to other chromatin-associated proteins has been investigated through a genetic interaction analysis. We scored for genetic modification of a domino wing margin phenotype through coexpression of RNAi directed against a set of previously characterized and more newly characterized chromatin-encoding loci. A set of other SWI2/SNF2 loci were also assayed for interaction with domino. Our results show that the majority of tested loci exhibit synergistic enhancement or suppression of the domino wing phenotype. Therefore, depression in domino function sensitizes the wing margin to alterations in the activity of numerous chromatin components. In several cases the genetic interactions are associated with changes in the level of cell death measured across the dorsal-ventral margin of the wing imaginal disc. These results highlight the broad realms of action of many chromatin proteins and suggest significant overlap with Domino function in fundamental cell processes, including cell proliferation, cell death and cell signaling.

A targeted genetic modifier screen links the SWI2/SNF2 protein domino to growth and autophagy genes in Drosophila melanogaster.

Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity.

Linking model systems to cancer therapeutics: the case of Mastermind.

Genetics, and more recently genomics, reveal striking conservation in the fundamental signaling pathways that underlie normal and aberrant cell processes. Consequently, various genetic model organisms are now attracting the interest of biomedical scientists who are focused on therapeutic approaches to human disease. There are now several examples of studies in which Drosophila seems likely to facilitate advances in potential therapies, and a recent report has demonstrated the utility of the fly model for understanding and treating human disease. Basic developmental genetic information first obtained in Drosophila was used to design a therapeutic block to oncogenic Notch signaling that was associated with leukemia in mice. The story of Notch signaling in Drosophila demonstrates the potential for standard Drosophila molecular genetics in developing therapeutic strategies that are relevant to human disease.

Targeted gain-of-function screening in Drosophila using GAL4-UAS and random transposon insertions.

Alterations in the activity level or temporal expression of key signalling genes elicit profound patterning effects during development. Consequently, gain-of-function genetic schemes that overexpress or misexpress such loci can identify novel candidates for functions essential for a developmental process. GAL4-Upstream Activating Sequence (UAS)-targeted regulation of gene expression in Drosophila has allowed rapid analyses of coding sequences for potential roles in specific tissues at particular developmental stages. GAL4 has also been combined with randomly mobilized transposons capable of UAS-directed misexpression or overexpression of flanking sequences. This combination has produced a genetic screening system that can uncover novel loci refractory to standard loss of function genetic approaches, such as redundant genes. Available libraries of strains with sequenced insertion sites can allow direct correlation of phenotypes to genetic function. These techniques have also been applied to genetic interaction screening, where a GAL4 driver and UAS-regulated insertion collection are combined with an extant mutant genotype. In this article, we summarize studies that have utilized GAL4-UAS overexpression or misexpression of random loci to screen for candidates involved in specific developmental processes.

A male-specific effect of dominant-negative Fos.

The transcription factor Fos contains a basic DNA binding domain combined with a leucine zipper (bZip). Expression of a truncated form of Fos in Drosophila that contains only the bZip region (Fos bZip) elicits phenotypes resembling fos mutations. These effects presumably derive from competition between wild-type and truncated forms for dimerization partners, with the truncation acting in a dominant-negative manner. We found that expression of Fos bZip elicits male-specific phenotypes. Moreover, genetic interactions occur between Fos bZip and mutations in loci encoding the X chromosome dosage compensation complex. Fos bZip effects are correlated with aberrant male X chromosome structure and depressed signaling through the X-linked Notch locus. Unexpectedly, the male-specific effects are not reproduced with Fos RNAi, suggesting that Fos bZip can be neomorphic in nature. These results provide insight into how mutations in bZip proteins can exhibit gain of function activity.

Investigating the genetic circuitry of mastermind in Drosophila, a notch signal effector.

Notch signaling regulates multiple developmental processes and is implicated in various human diseases. Through use of the Notch transcriptional co-activator mastermind, we conducted a screen for Notch signal modifiers using the Exelixis collection of insertional mutations, which affects approximately 50% of the Drosophila genome, recovering 160 genes never before associated with Notch, extending the previous roster of genes that interact functionally with the Notch pathway and mastermind. As the molecular identity for most recovered genes is known, gene ontology (GO) analysis was applied, grouping genes according to functional classifications. We identify novel Notch-associated GO categories, uncover nodes of integration between Notch and other signaling pathways, and unveil groups of modifiers that suggest the existence of Notch-independent mastermind functions, including a conserved ability to regulate Wnt signaling.

Insertional inactivation of the L13a ribosomal protein gene of Drosophila melanogaster identifies a new Minute locus.

We have utilized a transposable P element construct to scan the genome for modifiers of Drosophila Notch pathway phenotypes. From a collection of 2000 inserts we obtained two enhancers and one suppressor. Sequence analysis of the insertion regions demonstrated that two modifiers affect known components of the Notch pathway, whereas the third is an insert in the gene encoding ribosomal protein L13a at cytogenetic region 83B6-7. The insert in the RpL13A coding region creates a classic Minute mutation which enhances Notch pathway wing phenotypes. This report adds RpL13A to the list of Drosophila ribosomal protein genes that cause Minute phenotypes when mutated.

An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster.

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation.

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.