An Indel Polymorphism in the MtnA 3' Untranslated Region Is Associated with Gene Expression Variation and Local Adaptation in Drosophila melanogaster

Abstract

Insertions and deletions (indels) are a major source of genetic variation within species and may result in functional changes to coding or regulatory sequences. In this study we report that an indel polymorphism in the 3' untranslated region (UTR) of the metallothionein gene MtnA is associated with gene expression variation in natural populations of Drosophila melanogaster. A derived allele of MtnA with a 49-bp deletion in the 3' UTR segregates at high frequency in populations outside of sub-Saharan Africa. The frequency of the deletion increases with latitude across multiple continents and approaches 100% in northern Europe. Flies with the deletion have more than 4-fold higher MtnA expression than flies with the ancestral sequence. Using reporter gene constructs in transgenic flies, we show that the 3' UTR deletion significantly contributes to the observed expression difference. Population genetic analyses uncovered signatures of a selective sweep in the MtnA region within populations from northern Europe. We also find that the 3' UTR deletion is associated with increased oxidative stress tolerance. These results suggest that the 3' UTR deletion has been a target of selection for its ability to confer increased levels of MtnA expression in northern European populations, likely due to a local adaptive advantage of increased oxidative stress tolerance.

Fig 1. Structure of the MtnA locus.

Two transcripts that differ only in their 3’ UTRs have been annotated for MtnA (MtnA-RA and MtnA-RB). Dark blue boxes represent the UTRs with the arrowheads indicating the direction of transcription. Orange boxes represent the coding exons. The thin lines joining the coding exons represent introns. The location of the polymorphic indel, which is shared by both transcripts, is indicated by the red triangle. For the coding genes flanking MtnA (CG12947 and CG8500), only the whole gene model is shown.

Fig 2. Expression of metallothionein genes in the brain in two populations of D. melanogaster.

(A) Expression level of Mtn paralogs in the brain from RNA-seq data. Expression is reported in reads per kilobase per million mapped reads (RPKM). Only MtnA showed a significant difference in expression between a European (the Netherlands), shown in blue, and an African (Zimbabwe), shown in green, population (adjusted P < 10⁻⁷ in the RNA-seq analysis [12]). Expression of MtnC was not detected. (B) MtnA expression in the brains of European and African flies, as determined by qRT-PCR. The expression difference between populations is highly significant (t-test, P = 5x10^-5). In both panels, the error bars indicate the standard error of the mean.

Fig 3. Results of CNV assays.

Flies from Africa (Zimbabwe), shown in green, and Europe (the Netherlands), shown in blue, were tested for MtnA CNV. The close paralogs AttA and AttB were used as a positive control for multiple gene copies, while RpL32 was used as a single-copy reference.

Fig 4. Association between an indel polymorphism in the MtnA 3' UTR and gene expression variation.

(A) An indel (and a linked SNP marked in gray) in the MtnA 3' UTR are the only polymorphisms within the 6-kb MtnA region that show a large difference in frequency between an African and a European population of D. melanogaster. A comparison with three outgroup species, D. sechellia (Sec), D. simulans (Sim) and D. yakuba (Yak), indicated that the deletion is the derived variant. (B) MtnA expression in the brain and the gut of eight European (NL) lines, shown in blue, and eight African (ZK) lines, shown in green. The two European lines lacking the deletion, NL11 and NL15, show lower MtnA expression than those with the deletion.

Fig 5. Reporter gene constructs and their expression.

(A) The gray boxes represent the MtnA promoter, which is identical between the African and European alleles. The white boxes represent the GFP/lacZ reporter genes. The blue hatched box represents the MtnA 3’ UTR with the deletion. The green box represents the MtnA 3’ UTR with the additional 49 bp marked in red. The same color scheme applies to the bar plots. (B) The two versions of the GFP reporter gene differ significantly in expression in heads (t-test, P = 0.0019) and bodies (t-test, P = 0.0046), as assayed by qRT-PCR. (C) The two versions of the lacZ reporter gene differed significantly in expression in heads (t-test, P = 0.0006) and guts (t-test, P = 0.0001) as measured by β-galactosidase enzymatic activity. The error bars represent the standard error of the mean.

Fig 6. Expression of an MtnA-GFP reporter gene in the brain.

(A-C) GFP expression driven by the reporter gene construct with the ancestral MtnA 3’ UTR variant. (D-G) Higher magnification of the brain regions where GFP is expressed. AL: antennal lobe, MB: mushroom bodies, SOG: subesophageal ganglion, Lo: lobula, Me: medulla. In (G) the arrow indicates cells expressing GFP. Green: GFP, red: anti-disclarge, targeting general neuropil.

Fig 7. Evidence for positive selection at the MtnA locus.

(A) Watterson’s θ of D. melanogaster populations from Zimbabwe (ZK), the Netherlands (NL) and Sweden (SU) calculated in sliding windows of 500 bp with a step size of 250 bp. (B) F_st values for pairwise comparisons of ZK, NL and SU calculated in sliding windows of 500 bp with a step size of 250 bp. (C) Selective sweep (SweepFinder) analysis of the Netherlands population showing the composite likelihood ratio (CLR) statistic in sliding windows of 1000 bp. (D) Selective sweep (SweepFinder) analysis of the Swedish population showing the CLR statistic in sliding windows of 1000 bp. The black line indicates the 5% significance threshold calculated using the demographic model of Duchen et al. [5] for neutral simulations. The red line indicates the 5% significance threshold calculated using the demographic model of Werzner et al. [6] for neutral simulations and the gray dashed line indicates the 5% significance threshold using the model of Thornton and Andolfatto [35]. (E) Gene models for the 6-kb region analyzed. The gray highlighted region indicates the position of the 49-bp indel polymorphism in the MtnA 3’ UTR.

Fig 8. Proportional mortality after oxidative stress.

(A) RNAi-mediated MtnA knockdown (hatched lines) and control flies (solid lines). P-values are shown for within population/background comparisons. (B) Dutch (blue) and Malaysian (orange) flies with the deletion (hatched lines) and without the deletion (solid lines) in the MtnA 3’ UTR. Error bars indicate the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig 9. Proportional mortality after copper sulphate exposure.

(A,B) Copper tolerance in RNAi-mediated MtnA knockdown flies (white, RNAi-MtnA/Act5C-GAL4) and control flies expressing normal levels of MtnA (solid grey, control/Act5C-GAL4). (C) Male and (D) female flies from the Dutch (NL, blue) and the Malaysian (KL, orange) population with the deletion (hatched) and without the deletion (solid). P-values are shown for within population/background comparisons. Error bars indicate the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005.