Regulation of Drosophila Lifespan by bellwether Promoter Alleles

Abstract

Longevity varies among individuals, but how natural genetic variation contributes to variation in lifespan is poorly understood. Drosophila melanogaster presents an advantageous model system to explore the genetic underpinnings of longevity, since its generation time is brief and both the genetic background and rearing environment can be precisely controlled. The bellwether (blw) gene encodes the α subunit of mitochondrial ATP synthase. Since metabolic rate may influence lifespan, we investigated whether alternative haplotypes in the blw promoter affect lifespan when expressed in a co-isogenic background. We amplified 521 bp upstream promoter sequences containing alternative haplotypes and assessed promoter activity both in vitro and in vivo using a luciferase reporter system. The AG haplotype showed significantly greater expression of luciferase than the GT haplotype. We then overexpressed a blw cDNA construct driven by either the AG or GT haplotype promoter in transgenic flies and showed that the AG haplotype also results in greater blw cDNA expression and a significant decrease in lifespan relative to the GT promoter haplotype, in male flies only. Thus, our results show that naturally occurring regulatory variants of blw affect lifespan in a sex-specific manner.

Figure 1

Diagram of the structure of the D. melanogaster blw gene. The blw gene is located on Chr2R:22,799,099…22,802,180 [+] and generates a single transcript composed of a 5′-UTR (gray box), four exons (blue boxes) and a 3′-UTR (gray arrow) (Flybase.org). The two SNPs are located 150 bp [G/T] and 188 bp [A/G] upstream of the blw transcriptional start site (gray box). The location of PCR primers used to generate the blw-promoter or blw-cDNA are indicated by red arrows.

Figure 2

Relative strength of the putative promoter region of the blw gene. (a) Four different lengths of the blw-promoter region harboring the AG haplotype were cloned in the pGL3-basic vector and expressed in Drosophila S2 cells. Primer locations are indicated with respect to the transcriptional start site of the blw gene (see Fig. 1). Black lines represent the promoter length and the yellow arrow represents the luciferase reporter gene. (b) The 521 bp blw-promoter region harboring the AG, GG, AT and GT haplotypes were cloned in the pGL3-basic vector and expressed in Drosophila S2 cells. (c) The same promoters were expressed in flies using the Gal4-UAS binary expression system. For each experiment, protein lysates were extracted and luciferase expression was measured and normalized (relative light units; RLU). Gray bars represent the Drosophila S2 cells (Panels a and b). Female flies are represented by red bars and males by black bars (Panel c). Error bars are standard errors of the mean.

Figure 3

Effect of blw-cDNA overexpression on lifespan. (a) Normalized expression levels measured by quantitative RT-PCR of blw-cDNA when driven by the blw-AG and blw-GT promoters (red, females; black, males). (b) Median lifespan of flies that overexpress blw-cDNA from the blw-AG and blw-GT promoters (red, females; black, males). (c) Survival curves of male flies overexpressing blw-cDNA when driven by either the blw-AG (red) or blw-GT promoter (black). (d) Survival curves of female flies overexpressing blw-cDNA when driven by either the blw-AG (red) or blw-GT promoter (black). The control flies expressing the endogenous blw gene are shown by gray dotted lines.