MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans

Abstract

Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.

Figure 1. Pmir-34_2.2kb::gfp is expressed in various tissues during development of C. elegans and its expression is upregulated in dauers.

(A) Expression of Pmir-34_2.2kb::gfp reporter at different stages of animal development. (B) Changes in the expression pattern of Pmir-34_2.2kb::gfp from predauer to dauer stage and detailed expression pattern of Pmir-34_2.2kb::gfp at dauer stage. (C) Elevated Pmir-34_2.2kb::gfp expression is associated with the dauer larva gene expression program. i, WT, ii and iii, insulin-like signaling pathway mutants: daf-2(e1370), pdk-1(sa680), respectively. iv, v and vi, TGF-β signaling pathway mutants: daf-1(e1287), daf-3(mgDf90), daf-7(e1372). vii, DAF-12/NHR signaling: daf-9(e1406). viii, daf-16(mu86);daf-7(e1372). (D) Pmir-34_2.2kb::gfp expression levels are increase by various stress factors. (i) Three days old animal grown at 20 °C. (ii) One day old animal grown at 25 °C, (iii) Three days old animal starved for two days. (iv) Ten days old animal grown at 20 °C.

Figure 2. Native levels of mir-34 expression are required for correct morphogenesis of dauers and dauer survival.

(A) qPCR confirmation of miR-34 expression levels in the rescue and the overexpression strains. Error bars represent SD. N = 300, **P < 0.01, ***P < 0.0001, unpaired two-tailed t test. (B) Differences in body sizes of WT, mir-34(gk437), mir-34OE and rescue strains. Error bars represent SD. *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired two-tailed t test. (C) Differences in dauer survival of mir-34(gk437) and mir-34OE dauers at 20 °C and 25 °C. Error bars represent SD. 250 animals are scored per experiment, 3 replicate experiments per condition. Statistical significance of differences between N2 and other genetic backgrounds at a given temperature is calculated using unpaired two-tailed t test. **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) Differences in dauer formation rates of mir-34(gk437) dauers in daf-2(e1370) and daf-7(e1372) backgrounds. Error bars represent SD. 250 animals were assayed per condition, experiments were performed in triplicate. Statistical significance is calculated by two-tailed unpaired t test. **P < 0.01.

Figure 3. mir-34 is regulated by DAF-16 and targets daf-16.

(A) Upstream region of mir-34 pre-miRNA (in red) showing sequences used as promoters in Pmir34::gfp strains in blue and location of DAF-12, DAF-16 and PQM-1 modENCODE ChIP-seq peaks in black. (B) Expression patters of Pmir34::gfp strains with different promoter length. (C) Last exon and 3′UTR of daf-16 transcript (in blue), location of AGO-CLIP regions (in green) from Grosswendt et al. and highly-scoring MIRZA miR-34 target prediction (in red). (D). Detailed MIRZA alignment of the miR-34 target in daf-16 mRNA shown in panel C. (E) Expression of DAF-16::GFP is elevated with temperature in amphid neurons (indicated by arrows) in mir-34(gk437) mutants but not in wild-type animals. (F) Quantification of fluorescence density of DAF-16::GFP in wild-type and mir-34(gk437) background at 27.5 °C. Data are expressed in arbitrary fluorescence units. Error bars represent SD. Statistical significance is calculated by two-tailed unpaired t test. *P < 0.05.

Figure 4. Effects of mir-34 deletion or overexpression on gene expression under various conditions.

(A–E) Number of differentially expressed genes (EXPR) and enrichment statistics for several regulatory signals. DAF-16, PQM-1 and MDL-1, occupancy in promoter regions based on modENCODE ChIP-seq data for respective transcription factors. DBE, DAE, presence of the respective sequence motives in the promoter regions. MIRZA, miR-34 target predictions calculated by MIRZA. ALG – presence of AGO-CLIP regions from Grosswendt et al., overlapping miR-34 MIRZA predictions. The first number in each column relates to genes expressed higher in the first conditions, the second – to the genes expressed higher in the second condition. Up- and down- arrows represent significant over- and under-representation respectively. Statistical significance is calculated by Pearson’s Chi-squared test with Yates’ continuity correction and adjusted for multiple comparisons by Bonferroni correction. Adjusted P < 0.01 was considered as significant. ns – not significant. (F,G) Overexpression of miR-34 at 20 °C shifts gene expression profile in the same direction as temperature stress. (H) Genes that behave differently in WT and mir-34 mutants upon temperature stress.