Transcriptional Regulation of the Methuselah Gene by Dorsal Protein in Drosophila melanogaster

  • Kim, Hyukmin (Department of Biomedical Science and Institute of Bioscience and Biotechnology, College of Natural Sciences, Hallym University) ;
  • Kim, Jinsu (Department of Biomedical Science and Institute of Bioscience and Biotechnology, College of Natural Sciences, Hallym University) ;
  • Lee, Yoonsoo (Department of Biomedical Science and Institute of Bioscience and Biotechnology, College of Natural Sciences, Hallym University) ;
  • Yang, Jaeyeon (Department of Biomedical Science and Institute of Bioscience and Biotechnology, College of Natural Sciences, Hallym University) ;
  • Han, Kyuhyung (Department of Biomedical Science and Institute of Bioscience and Biotechnology, College of Natural Sciences, Hallym University)
  • Received : 2005.12.24
  • Accepted : 2006.01.24
  • Published : 2006.04.30


The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 ~ -272 (PE1) and +28 ~ +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth-luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein.


Supported by : Korean Ministry of Small and Medium Industry


  1. Han, K., Levine, M. S., and Levine, J. L. (1989) Synergistic activation and repression of transcription by Drosophila homeobox proteins. Cell 56, 573-583
  2. Valentine, S. A., Chen, G., Shandala, T., Fernandez, J., and Mische, S., et al. (1998) Dorsal-mediated repression requires the formation of a multiprotein repression complex at the ventral silencer. Mol. Cell. Biol. 18, 6584-6594
  3. Han, K. and Manley, J. L. (1993) Transcriptional repression by the Drosophila even-skipped protein: definition of a minimal repression domain. Genes Dev. 7, 491-503
  4. Lau, G.. W., Goumnerov, B. C., Walendziewicz, C. L., Hewitson, J., and Xiao, W., et al. (2003) The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa. Infect. Immun. 71, 4059-4066
  5. Rushlow, C. A., Han, K., Manley, J. L., and Levine, M. (1989) The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell 59, 1165-1177
  6. Pletcher, S. D., Macdonald, S. J., Marguerie, R., Certa, U., Stearns, S. C., et al. (2002) Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster. Curr. Biol. 12, 712-723
  7. Cai, H. N., Arnosti, D. N., and Levine, M. (1996) Long-range repression in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 93, 9309-9314
  8. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA
  9. Senger, K., Armstrong, G. W., Rowell, W. J., Kwan J. M., Markstein, M., et al. (2004) Immunity regulatory DNAs share common organizational features in Drosophila. Mol. Cell 13, 19-32
  10. Aigaki, T., Seong, K., and Matsuo, T. (2002) Longevity determination genes in Drosophila melanogaster. Mech. Ageing Dev. 123, 1531-1541
  11. Cvejic, S., Zhu, Z., Felice, S. J., Berman, Y., and Huang, X.-Y. (2004) The endogenous ligand stunted of the GPCR methuselah extends lifespan in Drosophila. Nat. Cell Biol. 6, 540-546
  12. Jiang, J., Cai, H., Zhou, Q., and Levine, M. (1993) Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors. EMBO J. 12, 3201-3209
  13. West Jr., A. P., Llamas, L. L., Snow, P. M., Benzer, S., and Bjorkman, P. J. (2001) Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan. Proc. Natl. Acad. Sci. USA 98, 3744-3749
  14. Manfruelli, P., Reichhart, J. M., Steward, R., Hoffmann, J. A., and Lemaitre, B. (1999) A mosaic analysis in Drosophila fat body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF. EMBO J. 18, 3380-3391
  15. Park, S.-Y., Heo, Y.-J., Kim, K.-S., and Cho, Y.-H. (2005) Drosophila melanogaster is susceptible to Vibrio cholerae infection. Mol. Cells 20, 409-415
  16. Song, W., Ranjan, R., Dawson-Scully, K., Bronk, P., Marin, L., et al. (2002) Presynaptic regulation of neurotransmission in Drosophila by the G protein-coupled receptor Methuselah. Neuron 36, 105-119
  17. Cho, Y.-S., Han, D., Baek, G., Park, S.-P., Yoon, S., et al. (1996) Versatile luciferase reporter plasmids for transcription studies in diverse eukaryotic cells. Korean J. Zool. 39, 378-386
  18. Han, K. (1996) An efficient DDAB-mediated transfection of Drosophila S2 cells. Nucleic Acids Res. 24, 4362-4363
  19. Helfand, S. L. and Rogina, B. (2003) Molecular genetics of aging in the fly: is this the end of the beginning? Bioassays 25, 134-141
  20. Tzou, P., De Gregorio, E., and Lemaitre, B. (2002) How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5, 102-110
  21. Brody, T. and Cravchik, A. (2000) Drosophila melanogaster G protein-coupled receptors. J. Cell Biol. 150, F83-F88
  22. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, M., and Hoffmann, J. A. (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983
  23. Lin, Y. J., Seroude, L., and Benzer, S. (1998) Extended life-span and stress resistance in the DrosophilaMutant Methuselah. Science 282, 943-946
  24. Walchli, S., Colinge, J., and Hooft van Huijsduijnen, R. (2000) MetaBlasts: tracing protein tyrosine phosphatase gene family roots from Man to Drosophila melanogaster and Caenorhabditis elegans genomes. Gene 253, 137-143