DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila*

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.

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-dDnmt2 GS12412 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 minimize the variation generated by differential rates of paraquat intake, 3-day-old flies were first starved for 6 h and then transferred to vials containing the parauqat-wetted filters. The survival was scored at 25°C in the dark.
RT 1 -PCR Analysis-The expression levels of different genes were assayed by semiquantitative PCR. For this, total RNAs were isolated from adult flies of the four different genotypes UAS-dDnmt2͗1͘/ϩ, UAS-dDnmt2͗1͘/ϩ;da-Ga14/ϩ, yw, and dDnmt2 GS12412 , respectively, and reverse transcribed for subscequent PCR reactions using DNA primers specific for different Drosophila genes. The PCR products were analyzed by agarose gel electrophoresis. Sequences of the primers are available upon request.
In contrast to the above is a P element line dDnmt2 GS12412 , in which a Gene Search vector (11) was inserted in exon 1 of the dDnmt2 gene. dDnmt2 GS12412 is homozygous viable and the level of its dDnmt2 RNA is at least 50% lower than the control yw line, which is the recipient stock of the GS insertion (compare lanes 1 and 2, Fig. 1B; see also Fig. 2A). While no obvious phenotypes could be seen during development, homozygous dDnmt2 GS12412 flies died more quickly than the heterozygotes with balancer. We followed up on this observation by examining the possible relationship between dDnmt2 gene expression and fly aging. As exemplified in Fig. 1B, life span measurements in comparison with yw showed that homozygous dDnmt2 GS12412 line lived, on the average, 27% shorter than yw flies, with the average life spans being 54 days and 75 days, respectively.
Longevity genes in the Drosopila also mediate increased resistance to various stresses (12). We have first carried out the thermotolerance measurement but found no difference of the average survival at 36°C, either between yw and homozygous dDnmt2 GS12412 or between UAS-dDnmt2/ϩ and UAS-dDnmt2/ ϩ;da-GAL4/ϩ. We then compared the different dDnmt2 lines  (n ϭ 134), respectively. C, flies were exposed to paraquat for examination of the sensitivity to oxidative stress. The survival percentages of different lines at 24 and 36 h of dieting are expressed by the bar histograph. described above with their respective controls for the resistance to starvation. In Caenorhabditis elegans, the mutant daf-2 has extensive fat accumulation and exhibits marked increase in longevity, suggesting the link between metabolism and longevity (13). In Drosophila, some long life flies like the mth and chico mutants also showed higher resistance to starvation (12,14). A 20% decrease of survival of the homozygous dDnmt2 GS12412 females relative to the yw females was found. Also, the dDnmt2-overexpressing transgenic flies, when compared with the UAS control lines, showed an increase of 20% in the females and 15% in the males (data not shown).
Oxidative damage to DNA and proteins has also been implicated in a variety of degenerative diseases. Accumulations of oxidative damages are associated with aging too. To test the resistance to oxidative stress, we fed flies with the free radical generator, paraquat. At 36 h after being fed with diet containing the paraquat, the dDnmt2-overexpressing flies exhibited higher resistance to oxidative stress than the controls (Fig. 1C), but no obvious difference was observed between dDnmt2 GS12412 and yw. Transgenic flies carrying extra copies of SOD and catalase genes, which function to eliminate reactive oxygen species, had an increased life span (see Ref. 15 and references therein). A null mutation in chico was homozygous viable, and the flies exhibited an increased life span as well as a higher level of the superoxide dismutase activity (14). Although our dDnmt2 overexpression flies showed more resistance to oxidative stress, the level of SOD gene expression in these flies was not altered (data not shown).
dDnmt2 could regulate the Drosophila life span by modulating the expression of a set of genes, which may or may not consist of genes previously demonstrated to affect the life spans of yeast, C. elegans, mice, and Drosophila (15)(16)(17)(18)(19). We have tested by RT-PCR the levels of expression of several of these "longevity" genes including InR, an insulin-like receptor; chico, an insulin-like receptor substrate protein; mth, a G-proteincoupled receptor; and SOD, the CuZn superoxide dismutase, as mentioned above. No significant differences of expression of these genes were found among the Drosophila lines analyzed in Fig. 1 (data not shown). Interestingly, however, we found that the expression levels of several small heat shock proteins (sHsp)-encoding genes (Hsp22, Hsp23, and Hsp26) were coordinately changed in flies with altered amounts dDnmt2 (Fig. 2). The levels were up-regulated, all by ϳ3-fold, in flies overexpressing dDnmt2 (compare lanes 2 with lanes 1 in D-F of Fig.  2), and they were down-regulated, by 3-, 2-, and 3-fold, respectively, in flies with decreased amount of dDnmt2 (compare lanes 4 with lanes 3 in D-F of Fig. 2). It should be noted here that life span analysis of a precise excision line of dDnmt2 GS12412 would conclusively demonstrate that the shortened life span of dDnmt2 GS12412 is not due to sporadic mutation(s) generated during construction of the original P element library. However, the parallel effects on the Dro-sophila by dDnmt2 (this study) and by Hsp22 (22,23) and our finding that the Hsp22 gene is regulated by dDnmt2 (Fig. 2) strongly suggest that the lengthening-shortening effects on the fly life span observed by us more likely resulted from the up-and-down-regulation of the dDnmt2 gene instead of sporadic mutation(s).
Small Hsps, like other Hsps, are molecular chaperones that are coordinately regulated by thermal stress and, more relevantly, they are involved in the aging processes (reviewed in Ref. 18). The levels of Hsp22, which is a mitochondrial protein, and the cytosolic Hsp26 are both higher in aged flies (20,21). More importantly, overexpression of Hsp22 and the two cytosolic small Hsps, Hsp23 and Hsp26, all increased the life span of Drosophila (11,18,22). Decrease of Hsp22 expression due to P element insertion led to shortened Drosophila life span (23). Also, overexpression of Hsp22 and Hsp26 increase the resistance to oxidative and heat stress (22,24). These studies all point to the beneficial effects of the small Hsps on longevity, presumably by preventing the aggregation of damaged and/or oxidized proteins (25). Our RT-PCR data in Fig. 2 suggest that one of the mechanisms for dDnmt2 to regulate the Drosophila life span is by acting upstream of the expression of the small Hsp genes, at least those encoding Hsp22, Hsp23, and Hsp26. Thus, the dDnmt2 gene has emerged as a newly discovered player in the regulation of Drosophila life span.
The regulation of the animal life span is modulated by multiple, and likely interconnected, cellular signaling pathways (see Refs. 12-18 and 23 and references therein). While the details of the molecular and cellular basis of dDnmt2 being a longevity gene awaits further investigation, our finding has uncovered a new function of the eukaryotic DNA methyltransferase gene family. Namely, a threshold level of expression of dDnmt2, a gene conserved among the flies, mammals, and yeast Schizosaccharomyces pombe (2,5,6), appears to be essential for the maintenance of a normal life span of Drosophila. While overexpression of exogenous mammalian DNA methyltransferases Dnmt1 and Dnmt3a in Drosophila caused fly lethality (26), elevation of the endogenous dDnmt2 level actually increased the life span of the flies, as shown in this study. Whether the same function is carried out by the mammalian Dnmt2 genes remains an intriguing question.