The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methylation programs in general.DNA methylation at cytosines of the vertebrate genomes participates in the control of several interlinked biological processes, e.g. gene expression, cell growth, genomic imprinting, X chromosome inactivation, and embryogenesis, which in turn are mediated through chromatin remodeling (1, 2). The vertebrate DNA methyltransferases, including Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, and Dnmt3L, are a family of proteins with highly conserved motifs in their carboxyl regions. Of these, Dnmt1 is the maintenance enzyme. Dnmt3a and Dnmt3b carry out the de novo methylation reaction. Unlike the above three enzymes, Dnmt2 lacks the N-terminal regulatory region (2). Although recent studies have demonstrated residual DNA methylation activities of the mammalian Dnmt2 proteins and the Drosophila ortholog dDnmt2 (3, 4), the latter of which is the single DNA methyltransferase responsible for genome methylation of the fruit flies (5-10), the functional role of the Dnmt2 proteins has remained unclear. We present evidence below that dDnmt2 is a regulator of the life span of Drosophila. EXPERIMENTAL PROCEDURES Drosophila Strains and Culture Conditions-dDnmt2GS12412 stock was a kind gift from Dr. T. Aigaki at Tokyo Metropolitan University. yw strain, the recipient stock of Gene Search (GS) insertion, was used as an internal control in the longevity experiment. UAS-dDnmt2 transgene was inserted in pUAST vector as described (10). The reference background for transgenic lines was w 1118 . UAS-dDnmt2 females were crossed with the da-GAL4 males or w 1118 males, and the life span of progeny was assessed. Flies were reared at 25°C and maintained in vials containing standard cornmeal agar medium. To monitor the life span, flies eclosed within 24 h were collected in single-sex groups of 20. They were then transferred to fresh medium and scored for survivorship once every 3 days until all of the flies died.Stress Resistance-For thermal stress test, 4-day-old flies were transferred to new vials, maintained, and scored for survivorship at 36°C. To examine the effect of starvation, flies were transferred to vials containing two 2.4-cm glass-fiber filter circles (Whatman) moisturized with 350 l of distilled water. The survivals were followed at 25°C. Distilled water was added to keep the filters moist during the test. Paraquat resistance was performed as follows. Whatman glass-fiber filter circles soaked with 350 l of 20 mM paraquat (Sigma) in 5% sucrose solution were placed in clean empty vials. To minimi...

Methylation of the vertebrate genomes at cytosines is known to be accomplished by the combined actions of proteins encoded by different cytosine-5 DNA methyltransferases or DNA MTases. These proteins include the de novo DNA MTases Dnmt3a and Dnmt3b, the maintenance DNA MTase Dnmt1, and their isomers (1-4). There are also other DNA MTase-like proteins expressed in the eukaryotic cells, but they are without well documented DNA methylation activities. These include the Dnmt3L (5) and Dnmt2 proteins.The Dnmt2 proteins are relatively shorter than Dnmt3a, Dnmt3b, or Dnmt1, and structurally they are similar to the bacteria dcm enzyme (6). The eukaryotic Dnmt2 protein family consists of the yeast pmt1 (7), the mammalian Dnmt2 including mouse mDnmt2 and human hDnmt2 (8, 9), and Drosophila dDnmt2 (10, 11). Of the Dnmt2 proteins known, pmt1 has been demonstrated to be enzymatically inactive due to amino acid change at a potential catalytic site (12). Sequence analysis showed that mDnmt2, hDnmt2, and dDnmt2 all contain the conserved DNA MTase motifs (8 -11). DNA methylation analysis of ES cells with homozygous knock-out of the mouse mD-NMT2 genes suggested that mDnmt2 protein might also be an inactive DNA MTase (8). Finally, there has been no report on the DNA methylation activity of the Drosophila dDnmt2 until very recently. By overexpression of dDnmt2 in Drosophila S2 cells and subsequent analysis of the S2 cell genome with the sodium bisulfite sequencing approach, it was shown that specific regions were anomalously methylated in comparison to S2 cells without overexpression of the dDnmt2 protein (13).To avoid potential side effects resulting from use of the long term-selected S2 cell culture in the above study, we have now examined the genome of transgenic Drosophila flies stably overexpressing dDnmt2. Interestingly, specific genomic regions of the transgenic flies were also found to be anomalously hypermethylated. To complement the fly analysis, we further carried out DNA transfection experiments to transiently express fly dDnmt2 or mouse mDnmt2 in S2 cells. As shown below, dDnmt2 as well as mDnmt2 are capable of methylating cytosines of a cotransfected plasmid. The conservation of the enzymatically active Dnmt2 proteins from mammals to the flies suggests that this DNA MTase subfamily likely carries out important and to-be-identified function(s). EXPERIMENTAL PROCEDURESPlasmid Constructs-For transgenic fly work, the dDNMT2 cDNA was released from pGEM(1)-dDNMT2 with PacI-NcoI and blunt ended. This dDNMT2 fragment, which contains bp 6964 -7024 linked to 7074 -8051 of the dDNMT2 gene, was cloned at the EcoRI site of the polylinker of pUAST (14), and the resulting plasmid, pUAST-dDNMT2, was used for germ line transformation. For transient transfection, pAC5.1/V5-HisA vector (Invitrogen) containing Drosophila actin promoter was digested with KpnI, blunt ended, and redigested with EcoRI. pSG424 plasmid (15) was cut with BglII, blunt ended, and the GAL4 DNA binding domain-containing fragment was released by EcoRI digestion. Th...

Like vertebrates, the genome of Drosophila melanogaster also contains methylated cytosines. However, the enzyme(s) responsible for this methylation has been elusive. By DNA transfection and sodium bisulfite sequencing, we show here that overexpression of dDnmt2, which is the only expressed and cloned Drosophila protein consisting of motifs conserved among the DNA cytosine methyltransferases, results in genomic DNA methylation of Drosophila S2 cells. The data provide the first evidence for dDnmt2 being one candidate gene encoding the Drosophila DNA methyltransferase(s).