The epigenetic regulator G9a attenuates stress-induced resistance and metabolic transcriptional programs across different stressors and species

Abstract

Background: Resistance and tolerance are two coexisting defense strategies for fighting infections. Resistance is mediated by signaling pathways that induce transcriptional activation of resistance factors that directly eliminate the pathogen. Tolerance refers to adaptations that limit the health impact of a given pathogen burden, without targeting the infectious agent. The key players governing immune tolerance are largely unknown. In Drosophila, the histone H3 lysine 9 (H3K9) methyltransferase G9a was shown to mediate tolerance to virus infection and oxidative stress (OS), suggesting that abiotic stresses like OS may also evoke tolerance mechanisms. In response to both virus and OS, stress resistance genes were overinduced in Drosophila G9a mutants, suggesting an intact but overactive stress response. We recently demonstrated that G9a promotes tolerance to OS by maintaining metabolic homeostasis and safeguarding energy availability, but it remained unclear if this mechanism also applies to viral infection, or is conserved in other species and stress responses. To address these questions, we analyzed publicly available datasets from Drosophila, mouse, and human in which global gene expression levels were measured in G9a-depleted conditions and controls at different time points upon stress exposure.

Results: In all investigated datasets, G9a attenuates the transcriptional stress responses that confer resistance against the encountered stressor. Comparative analysis of conserved G9a-dependent stress response genes suggests that G9a is an intimate part of the design principles of stress resistance, buffering the induction of promiscuous stress signaling pathways and stress-specific resistance factors. Importantly, we find stress-dependent downregulation of metabolic genes to also be dependent on G9a across all of the tested datasets.

Conclusions: These results suggest that G9a sets the balance between activation of resistance genes and maintaining metabolic homeostasis, thereby ensuring optimal organismal performance during exposure to diverse types of stress across different species. We therefore propose G9a as a potentially conserved master regulator underlying the widely important, yet poorly understood, concept of stress tolerance.

Keywords: Drosophila; G9a; Mammalian cells; Metabolism; Resistance; Stress response; Tolerance; Transcription.

Fig. 1

Stress-induced gene expression changes are increased in G9a-depleted conditions. Bar graph showing the number of differentially expressed genes identified in pairwise comparisons of mRNA levels before and after stress exposure in G9a-depleted conditions (orange) and controls (beige). Differentially expressed genes were defined by an adjusted p value < 0.05, and a fold change > 1.2 up (normal fill) or down (striped)

Fig. 2

G9a attenuates stress-induced gene expression changes in different species under different types of stress. Boxplots and heat maps showing log2 fold changes of G9a-dependent stress-responsive genes across all five published datasets. Stress-dependent gene expression changes tend to be increased in magnitude in G9a-depleted conditions, being either overinduced or overrepressed after stress exposure. The numerical data depicted in this figure can be found in Table S1

Fig. 3

Stress response genes are overinduced and metabolic genes are overrepressed in G9a-depleted conditions. a, b Heatmaps showing p values for GO enrichment highlighted in light to dark blue. Not significant terms are highlighted gray. Columns show overinduced (column 1–5, annotated in red) and overrepressed (column 6–10, annotated in purple) gene groups from each dataset. Row colors indicate -log10 of p value (false discovery rate) for enrichment of the indicated GO terms in each dataset. Here we show all selected common GO terms subdivided into functional groups of a stress-related terms: stress response, immune response, cellular signaling and transcription, and additional biological processes; and b metabolic terms: metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism

Fig. 4

Identification of orthologous genes that are commonly regulated by G9a in different species under different types of stress. a, b Heatmaps indicating pairwise overlap of genes between the five datasets for the a overinduced and b overrepressed gene groups. The total number of fly genes, or fly orthologs of mouse and human genes, is indicated below the dataset name. Overlap statistics on each tile connecting two datasets include hypergeometric p value for the enrichment, number of overlapping genes, fold enrichment, and percentage of overlap. The similarity between two datasets is represented by the Jaccard index as a color gradient in the tile. c, d Venn diagram showing overlaps between datasets for the c overinduced and d overrepressed genes groups. Total number of fly genes, or fly orthologs of mouse and human genes, is indicated below the dataset name. Overlap between two or more groups is highlighted in blue and unique genes in each dataset are indicated in beige

Fig. 5

G9a buffers the activation of stress-specific resistance genes. We selected genes that were uniquely overinduced in only one of the five G9a-depleted datasets and were annotated with the specific GO terms “response to oxidative stress,” “immune response,” and “response to hypoxia.” Based on published evidence, we classified the function of each gene in the stress response in the following categories: detection (proteins that are involved in sensing the stress), resistance (proteins are directly involved in eliminating the stress), gene regulation (transcription factors, translation regulators, chromatin regulators, and cofactors that activate or repress a stress-responsive transcription factor), signaling (any molecule involved in a signal transduction pathway), and unknown (annotated in stress response based on a mutant phenotypes or transcriptional response without mechanistic understanding)

Fig. 6

G9a attenuates a network of stress-activated genes in different species under different types of stress. a Protein interaction networks of overinduced overlap genes, as generated using the STRING app in Cytoscape. Of the 740 overinduced overlap genes, 237 are connected to at least one other gene in the group. The color-code indicates enriched KEGG terms associated with the networks, as indicated in b. b KEGG pathway enrichment of genes within the overinduced networks

Fig. 7

G9a attenuates a network of repressed genes in different species under different types of stress. a Protein interaction networks of overrepressed overlap genes, as generated using the STRING app in Cytoscape. Of the 841 overrepressed overlap genes, 364 are connected to at least one other gene in the group. The color-code indicates enriched KEGG terms associated with the networks, as indicated in b. b KEGG pathway enrichment of genes within the overrepressed networks