HomeData from the Gustin lab show that overexpression of the broad-complex (BRC) gene induces stress resistance and longer lifespan in adult Drosophila melanogaster fruit flies. The gonad is an important regulator of stress resistance and longevity in several types of organisms (3, 10). BRC, a key mediator of the Drosophila sex steroid hormone ecdysone, is normally expressed in the somatic cells of the gonad (2, 4, 15). Thus, we are very excited about the possibility that BRC mediates the known control of longevity by the sex steroid hormone ecdysone in flies (14).
The proposed analysis will tackle the still-unanswered question of how BRC regulates the stress resistance of flies. The BRC gene produces several different mRNAs, each encoding different transcription factors with a shared "core" domain (1, 5). The "Z1" form is of particular interest because its expression level most closely corresponds to the stress resistance of the fly. One possibility is therefore that BRC Z1 mediates induction in adult flies of stress response genes such as those encoding heat shock proteins (6,7) or stress hormones (8). To address this possibility, we have obtained a fly line containing a transgene that allows controlled over-expression of just BRC Z1 mRNA (1). In addition, we will shortly generate a transgenic fly line with controlled expression of double stranded RNA (dsRNA) specific to BRC Z1 mRNA. Expression of the dsRNA causes degradation of the specific mRNA in vivo, a phenomenon called RNA interference (9, 11).
In overview, the goal of this proposed microarray analysis will be to determine the effect of BRC Z1 mRNA overexpression or under-expression on levels of mRNA from other genes in adult flies, with particular attention to effects on mRNA expression for various stress resistance genes.
Array 1 procedure: analysis of the response to BRC overexpression. The HS-BRC Z1 fly line and the genetically matched control w1118 line will each be exposed to a 15 minute heat shock at 37 °C and then a 3-hr recovery at 25 0C. Flies (40 each) will then be frozen at -80°C in TRIzol, RNA extracted from pulverized frozen flies, RNA from each line separately labeled and then mixed together for analysis of hybridization to a single genomic DNA array. The heat shock induces expression of BRC-Z1 mRNA from the transgene which contains the heat shock promoter fused to the BRC Z1 cDNA (1).
Array 2 procedure: analysis of the response to BRC underexpression. The tub-PSwitch/UAS-BRC Z1 RNAi double-transgenic flies will be briefly starved and then grown for 6 hr on medium containing either the PSwitch inducer RU-486 or its carrier, ethanol. The Pswitch transcription factor, a modified version of the progesterone receptor, is ubiquitously expressed under control of the tubulin promoter and induced to become active by the progesterone analog RU-486 (12, 13). The double-transgenic fly line expresses BRCZ1 dsRNA under control of the active PSwitch protein. Flies treated with RU-486 or ethanol (50 each) will then be frozen on dry ice, RNA extracted from pulverized frozen flies, RNA from each line separately labeled and then mixed together for analysis of hybridization to a single genomic DNA array.
References
1. Bayer, C. A., L. von Kalm, and J. W. Fristrom. 1997. Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Dev Biol 187:267-82.
2. Buszczak, M., M. R. Freeman, J. R. Carlson, M. Bender, L. Cooley, and W. A. Segraves. 1999. Ecdysone response genes govern egg chamber development during mid- oogenesis in Drosophila. Development 126:4581-9.
3. Cargill, S. L., J. R. Carey, H. G. Muller, and G. Anderson. 2003. Age of ovary determines remaining life expectancy in old ovariectomized mice. Aging Cell 2:185-90.
4. Deng, W. M., and M. Bownes. 1997. Two signalling pathways specify localised expression of the Broad- Complex in Drosophila eggshell patterning and morphogenesis. Development 124:4639-47.
5. DiBello, P. R., D. A. Withers, C. A. Bayer, J. W. Fristrom, and G. M. Guild. 1991. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics 129:385-97.
6. Dubrovsky, E. B., G. Dretzen, and M. Bellard. 1994. The Drosophila broad-complex regulates developmental changes in transcription and chromatin structure of the 67B heat-shock gene cluster. J Mol Biol 241:353-62.
7. Dubrovsky, E. B., G. Dretzen, and E. M. Berger. 1996. The Broad-Complex gene is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene. Mol Cell Biol 16:6542-52.
8. Ekengren, S., Y. Tryselius, M. S. Dushay, G. Liu, H. Steiner, and D. Hultmark. 2001. A humoral stress response in Drosophila. Curr Biol 11:714-8.
9. Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-11.
10. Hsin, H., and C. Kenyon. 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399:362-6.
11. Roignant, J. Y., C. Carre, B. Mugat, D. Szymczak, J. A. Lepesant, and C. Antoniewski. 2003. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. Rna 9:299-308.
12. Roman, G., and R. L. Davis. 2002. Conditional expression of UAS-transgenes in the adult eye with a new gene-switch vector system. Genesis 34:127-31.
13. Roman, G., K. Endo, L. Zong, and R. L. Davis. 2001. P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster. Proc Natl Acad Sci U S A 98:12602-7.
14. Simon, A. F., C. Shih, A. Mack, and S. Benzer. 2003. Steroid control of longevity in Drosophila melanogaster. Science 299:1407-10.
15. Tzolovsky, G., W. M. Deng, T. Schlitt, and M. Bownes. 1999. The function of the broad-complex during Drosophila melanogaster oogenesis. Genetics 153:1371-83.
Copyright, Acknowledgements,
and Intended Use
Created by B. Beason (bbeason@rice.edu),
Rice University, 29 February 2004
Updated 20 July 2006
