CHARACTERIZATION OF THE MENIN AND TIP60 INTERACTION IN DROSOPHILA MELANOGASTER

Abstract

<p>Drosophila melanogaster (fruit fly) has been used as a model organism for almost a century. Drosophila shares key molecular pathways and regulatory proteins with humans, making it a useful organism to study human diseases. The disruption of these pathways often leads to similar defects in flies and humans. For example, the Fragile X mental retardation syndrome is caused by the disruption of FMR1, leading to intellectual and physical impairment from defects in synaptic transmission . Flies lacking dfmrl show behavioural defects similar to humans, and also have impaired motor control. The Ras pathway is also well studied using the Drosophila model system and mutations in Ras1 show defects in cell proliferation in flies and humans. Multiple endocrine neoplasia type 1 is another syndrome that can be studied using Drosophila as a model organism.</p> <p>Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome predominantly characterized by tumour formation in various endocrine glands. Heterozygous patients for MENl develop the disease following a loss of the wild type allele (loss of heterozygosity). Menin is encoded by MEN1, and has no obvious sequence homology to other proteins. In order to gain some insight on the role of menin, several studies have looked for proteins interacting with menin. Using a yeast two-hybrid screen, our laboratory identified Enhancer of Polycomb E(Pc), a component of the Tip60 histone acetyltransferase complex as a candidate, novel menin interacting protein. Closer investigation revealed dTip60 as an interacting partner of Menin in Drosophila S2 cells.</p> <p>Menin was found to interact with dTip60 in S2 cells under optimal growing conditions and after lhr of heat shock. However, co-immunoprecipitation after stress by Alfa-irradiation did not reveal an interaction. Additionally, the pattern of expression and role of dTip60 was examined in wild type Oregon R flies and in flies expressing RNAi against dTip60. Heat shock lethality experiments were conducted to compare Tip60 RNAi flies to controls . dTip60 did not appear to playa role in the stress response at the larval developmental stages. The Tip60 RNAi flies showed robust expression of dTip60 throughout embryogenesis; therefore the maternal contribution of dTip60 was also investigated. The maternal contribution of dTip60 was significantly reduced in embryos from females harbouring the UAS-dTip60 RNAi and daGAL4 constructs. This can be a valuable tool for future experiments with embryos into the function of dTip60.</p> <p>Further experiments must be conducted to determine whether a dynamic interaction occurs after v-irradiation using closer kinetics in S2 cells. Also, experiments using wild type flies showed that dTip60 is developmentally regulated . dTip60 is required for development since Tip60 RNAi flies do not survive past the pupal stage. Increased lethality is not seen in Tip60 RNAi flies compared to controls when subjected to heat shock, suggesting that dTip60 does not playa role in stress response at this time. A v-irradiation lethality experiment should be conducted to determine if a complex containing menin and dTip60 is required for DNA damage repair. Lastly, to determine whether menin and dTip60 have a role in the same pathway, stress experiments must be conducted using Mnnr1^(-/-) flies as a comparison to phenotypes seen in Tip60 RNAi flies.</p>