Mitochondria as environments for the nuclear genome in Drosophila: mitonuclear G×G×E

Abstract

Mitochondria evolved from a union of microbial cells belonging to distinct lineages that were likely anaerobic. The evolution of eukaryotes required a massive reorganization of the 2 genomes and eventual adaptation to aerobic environments. The nutrients and oxygen that sustain eukaryotic metabolism today are processed in mitochondria through coordinated expression of 37 mitochondrial genes and over 1000 nuclear genes. This puts mitochondria at the nexus of gene-by-gene (G×G) and gene-by-environment (G×E) interactions that sustain life. Here we use a Drosophila model of mitonuclear genetic interactions to explore the notion that mitochondria are environments for the nuclear genome, and vice versa. We construct factorial combinations of mtDNA and nuclear chromosomes to test for epistatic interactions (G×G), and expose these mitonuclear genotypes to altered dietary environments to examine G×E interactions. We use development time and genome-wide RNAseq analyses to assess the relative contributions of mtDNA, nuclear chromosomes, and environmental effects on these traits (mitonuclear G×G×E). We show that the nuclear transcriptional response to alternative mitochondrial "environments" (G×G) has significant overlap with the transcriptional response of mitonuclear genotypes to altered dietary environments. These analyses point to specific transcription factors (e.g., giant) that mediated these interactions, and identified coexpressed modules of genes that may account for the overlap in differentially expressed genes. Roughly 20% of the transcriptome includes G×G genes that are concordant with G×E genes, suggesting that mitonuclear interactions are part of an organism's environment.

Keywords: coevolution; epistasis; gene expression module.

Figure 1.

(A) Experimental design for the construction of mitonuclear genotypes from different mtDNAs paired with specific DGRP nuclear genotypes. Three mtDNAs from geographic samples of D. melanogaster (OreR, Zim53, Aut) and 3 mtDNAs from the 3 distinct mtDNA haplotypes in D. simulans (siI, siII, siIII represented by “ma1”) were placed on each of 12 DGRP nuclear genomic backgrounds. The top 3 rows of the grid represent “Home Team” mitonuclear combinations, of D. melanogaster mtDNAs on D. melanogaster nuclear backgrounds (blue mtDNAs with blue nuclear chromosomes); the bottom 3 rows represent “Away Team” mitonuclear combinations of foreign D. simulans mtDNAs on D. melanogaster nuclear backgrounds (red mtDNAs with blue chromosomes). The phylogeny at the left shows the number of amino acid changes between the various lineages of the phylogeny. A prediction of the mitonuclear coadaptation hypothesis is that the Away Team combinations would show greater dysfunction due to mitonuclear incompatibilities resulting from ~2.5 million years of independent mutation in the respective genomes. (B) Experimental treatment showing the subset of mitonuclear genotypes used in the diet exposure and RNAseq experiment. The 4 mitonuclear genotypes developed from egg to adult on Lab Food. At age 4 days, replicate cultures of 30 single sex adults were transferred to fresh Lab Food for 2 days. At time = 0, replicates were frozen as controls, and remaining replicates were transferred to each of 2 alternative diets for 60 and for 120 min: Y = High P:C and to S = Low P:C for Yeast and Sugar respectively, and then frozen for later RNA extraction.

Figure 2.

G×G×E for development time in mitonuclear genotypes. The upper left depicts the 6×12, mito × nuclear genotypes placed on 4 different diets. The larger graph plots the development time for each mitonuclear genotype x diet combination, color coded by diet type. The insets show development times for mtDNAs on 2 specific nuclear backgrounds (DGRP-315 and DGRP-714) on each of the 4 diets.

Figure 3.

Transcripts affected by mitonuclear G×G overlap with G×E transcripts.(A) Statistical models for assessing G×G and G×E effects on nuclear transcripts as a function of mtDNA (Mito), nuclearDNA (Nuclear), and Environment (Enviro). The interaction terms in red represent the focal statistic for comparing G×G and G×E effects. (B) Heatmap of coexpressed transcripts: rows are transcripts, columns are nuclear, mtDNA and diet and time treatments, as specified in the column headers. The C, Y, and S columns refer to Control Lab Food (C), or food with high Yeast or high Sugar, equivalent to High P:C or Low P:C food, respectively. The Time column headers refer to Control Food (0), or 1 (1) or 2 (2) h on the alternative food. (C) Venn diagrams showing overlap of G×G and G×E for females on the High P:C diet (top) or Low P:C diet (bottom), relative to lab food. (D) Permutation tests showing that observed data show greater enrichment of overlap (colored curves) compared to a randomization of the G×G and G×E gene lists. Data from 4 data partitions are shown: Females on High P:C and Low P:C and Males on High P:C and Low P:C diets, compared to Lab Food. The Venn diagram for males is in Supplementary Figure S1.

Figure 4.

MtDNA genotype alters the transcriptional response to refeeding in a rapamycin-dependent manner. (A) Modules of genes that show inverted time course of expression for the “away team” genotype sm21;OreR when exposed to rapamycin (compare Rapa time courses for OreR;OreR vs. sm21:OreR). The transcripts in this module are associated with oxidative phosphorylation, carbohydrate metabolism, and nucleotide metabolism as shown in the heat maps below the time-course plots. (B) Cartoon showing that 2 distinct mitonuclear expression experiments both point to the transcription factor giant as a shared regulator of gene expression. (C) Network diagram illustrating the association between the giant transcription factor (left, red node) and the Dref transcription factor (right, blue node) and the respective transcripts that are associated with these transcription factors. Purple nodes are transcripts where analyses infer transcriptional modification from both giant and Dref.