Epigenetic mechanisms modulate differences in Drosophila foraging behavior

Abstract

Little is known about how genetic variation and epigenetic marks interact to shape differences in behavior. The foraging (for) gene regulates behavioral differences between the rover and sitter Drosophila melanogaster strains, but the molecular mechanisms through which it does so have remained elusive. We show that the epigenetic regulator G9a interacts with for to regulate strain-specific adult foraging behavior through allele-specific histone methylation of a for promoter (pr4). Rovers have higher pr4 H3K9me dimethylation, lower pr4 RNA expression, and higher foraging scores than sitters. The rover-sitter differences disappear in the presence of G9a null mutant alleles, showing that G9a is necessary for these differences. Furthermore, rover foraging scores can be phenocopied by transgenically reducing pr4 expression in sitters. This compelling evidence shows that genetic variation can interact with an epigenetic modifier to produce differences in gene expression, establishing a behavioral polymorphism in Drosophila.

Keywords: Drosophila melanogaster; behavior; epigenetics; foraging; histone methylation.

Fig. 1.

foraging (for) interacts with G9a to mediate differences in adult feeding behavior. (A) The for gene model. The four transcription start sites (pr1–4) are marked with green arrows, exons are in gray boxes, and introns are shown as black lines. G9a-associated methylation sites are shown in purple. (B) Foraging arena schematic. (C) Rovers find significantly more sucrose drops than sitters in 5 min (t₅₈ = −6.829; P < 0.001) and 10 min (t₅₈ = −8.523; P < 0.001). (D) Rovers with G9a WT find significantly more sucrose drops than sitters with G9a WT during a 5-min test [F_(3,116) = 10.58; P < 0.001], but this difference disappears in G9a null flies (P = 0.751). (E) Rovers with G9a WT find significantly more sucrose drops than sitters with G9a WT during a 10-min test [F_(3,116) = 10.88; P = 0.004], but this difference disappears in G9a null flies (P = 0.678). n = 20/strain.

Fig. S1.

G9a affects starvation resistance significantly more in sitters than in rovers. Here 5- to 6-d-old adult females were transferred to agar vials with a water source in groups of 10, and the number of dead flies was scored every 6 h until the last fly died. While G9a nulls show increased starvation resistance in both rovers and sitters (Kaplan–Meier survival statistic, S₃ = 214.225; P < 0.001), the G9a-mediated response in starvation resistance is significantly larger in sitters (S₃ = 116.167; P = 0.00) than in rovers (S₁ = 62.133; P = 1.3E-14); n = 10.

Fig. 2.

The for promoters are differentially expressed and show G9a-dependent methylation differences. (A) Rovers and sitters do not differ in for pr1 expression [F_(3,11) = 3.96; P = 0.053]. (B) Rovers with G9a WT have significantly less pr2 expression than sitters with G9a WT [F_(3,11) = 42.39; P < 0.001], and this difference is not G9a-dependent as it is maintained in G9a nulls (P < 0.001). (C) Rovers and sitters do not differ in for P1/3 expression [F_(3,11) = 2.24; P = 0.161]. (D) Rovers with G9a WT have significantly less pr4 expression than sitters with G9a WT [F_(3,11) = 10.62; P < 0.003], and this difference is G9a-dependent, as it disappears in G9a nulls (P = 0.2). (E) Rovers with G9a WT have significantly more for pr1 H3K9me2 than sitters with G9a WT [F_(3,15) = 6.59; P = 0.007]. (F) Rovers with G9a WT have significantly less pr2 H3K9me2 than sitters with G9a WT [F_(3,15) = 68.86; P < 0.001], and this difference is not G9a-dependent, as it is maintained in G9a nulls (P < 0.001). (G) Rovers and sitters do not differ in for pr3 H3K9me2. (H) Rovers with G9a WT have significantly more pr4 H3K9me2 than sitters with G9a WT [F_(3,15) = 8.62; P = 0.002], and this difference is G9a-dependent, as it disappears in G9a nulls (P = 0.476). n = 3 for qRT-PCR and n = 4 for ChIP-qPCR with 20 adult mated females/biological replicate. *0.05 > P > 0.01; **0.01 > P > 0.001; ***P > 0.001.

Fig. S2.

Relative expression (qRT-PCR) of the three H3K9me2 histone methyltransferases in Drosophila, G9a, egg, and SU(VAR)3–9. (A) G9a expression does not differ between rovers and sitters. (B) egg expression shows no significant differences between strains and thus no association with G9a or for. (C) SU(VAR)3–9 expression differs between strains [F_(3,11) = 17.91; P = 0.001], in a pattern suggesting that for and G9a interact to regulate SU(VAR)3–9 expression, as opposed to for being regulated by SU(VAR)3–9. n = 3 with 20 adult mated females/biological replicate. *0.05 > P > 0.01; **0.01 > P > 0.001; ***P > 0.001.

Fig. 3.

Rover–sitter foraging behavior is directly regulated by pr4 expression levels. (A) Representative images of rover and sitter foraging paths with position coordinates over 10 min plotted as a scatterplot. (B) There are significant differences in foraging behavior among rovers, sitters, and rover–sitter heterozygotes [F_(4,119) = 11.09; P < 0.001], driven by G9a. Rovers with WT G9a forage significantly more in 10 min than sitters with WT G9a (P < 0.001). rover–sitter heterozygotes with WT G9a forage significantly more than sitters with WT G9a (P = 0.017), and are not significantly different from rovers with WT G9a (P = 0.138). Rovers, sitters, and rover–sitter heterozygotes with G9a null show no differences in foraging behavior. (C) Reducing pr4 expression by driving pr4-RNAi with the da-GAL4 driver significantly affects foraging behavior [F_(4,94) = 13.49; P < 0.001]. pr4 RNAi expression significantly increases sitter foraging behavior compared with controls (P = 0.002 compared with UAS control and P = 0.014 compared with GAL4 control), and does not significantly alter rover behavior (P = 0.719). (D) Reducing pr4 expression by driving pr4-RNAi with the da-GAL4 driver significantly affects the proportion of time spent in the interior food-containing area of the arena [F_(4,70) = 6.88; P < 0.001]. (E) Reducing pr4 expression by driving pr4-RNAi with the da-GAL4 driver does not affect the total distance traveled during foraging [F_(4,68) = 4.16; P = 0.005]. n = 20 for all tests. *0.05 > P > 0.01; **0.01 > P > 0.001.

Fig. S3.

Relative expression (qRT-PCR) of overall for expression and of RNAi knockdown using the for-RNAi for pr4 in whole flies. (A) There are no significant genotype differences in overall for expression in whole flies [F_(3,11) = 0.57; P = 0.653]. (B–F) Our RNAi line generated to target pr4 transcripts knocks down only pr4 transcripts. (B) Expression of this RNAi has no significant effect on overall for expression in rovers and sitters compared with controls. (C) Expression of this RNAi has no significant effect on for pr1 expression in rovers and sitters compared with controls. (D) Expression of this RNAi has no significant effect on for pr2 expression in rovers and sitters compared with controls. (E) Expression of this RNAi has no significant effect on expression of the P1/3 class of transcripts. (F) Expression of this RNAi has a significant effect on overall for pr4 expression in rovers and sitters compared with controls [t₃ = 14.339, P < 0.001, for rovers compared with rover control; F_(2,11) = 6.087, P = 0.021, for sitters compared with sitter controls]. n = 4, with 20 adult virgin females/biological replicate.

Fig. 4.

The rover–sitter difference in for pr4 expression arises from the brain and ovaries. (A) Rovers with G9a WT have significantly less pr4 expression in the brain than sitters with G9a WT [F_(3,11) = 32; P < 0.001], and this difference is G9a-dependent, as it disappears in G9a nulls (P = 0.19). (B) Rovers and sitters do not differ in for pr4 expression in the gut [F_(3,11) = 0.13; P = 0.937]. (C) Rovers with G9a WT have significantly less pr4 expression in the ovaries than sitters with G9a WT [F_(3,11) = 10.34; P = 0.004], and this difference is G9a-dependent, as it disappears in G9a nulls (P = 0.19). (D) Rovers and sitters with G9a WT do not differ in for pr4 expression in the carcass [F_(3,11) = 8.03; P = 0.242], but rovers with G9a null have significantly less pr4 expression in the carcass than sitters with G9a WT (P = 0.014). (E) Rovers and sitters differ in one SNP in a 0.1-kb region upstream of the pr4 transcription start site. For qRT-PCR, n = 3, with 20 adult mated female tissues/biological replicate for all tissues. *0.05 > P > 0.01; **0.01 > P > 0.001; ***P > 0.001.

Fig. S4.

ChIP-qPCR for high and low H3K9me2-methylated control genes. (A) moca-cyp, used as a negative control, showed low methylation (<3%) in all strains. (B and C) 2cta (B) and 2chi (C), used as positive controls, show high methylation (40–50%) in all strains, with no significant differences among strains. n = 4 with 20 adult mated females/biological replicate.