Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans

Abstract

DNA damage causally contributes to aging and age-related diseases. Mutations in nucleotide excision repair (NER) genes cause highly complex congenital syndromes characterized by growth retardation, cancer susceptibility, and accelerated aging in humans. Orthologous mutations in Caenorhabditis elegans lead to growth delay, genome instability, and accelerated functional decline, thus allowing investigation of the consequences of persistent DNA damage during development and aging in a simple metazoan model. Here, we conducted proteome, lipidome, and phosphoproteome analysis of NER-deficient animals in response to UV treatment to gain comprehensive insights into the full range of physiological adaptations to unrepaired DNA damage. We derive metabolic changes indicative of a tissue maintenance program and implicate an autophagy-mediated proteostatic response. We assign central roles for the insulin-, EGF-, and AMPK-like signaling pathways in orchestrating the adaptive response to DNA damage. Our results provide insights into the DNA damage responses in the organismal context.

Keywords: Caenorhabditis elegans; DNA damage response; DNA repair; aging; lipidomics; nucleotide excision repair; proteomics.

Graphical abstract

Figure 1

Proteome Analysis of the DNA Damage Response in NER Deficient C. elegans (A) Experimental workflow. xpc-1;csb-1 double-mutant L1 larvae were treated with 100 mJ/cm² UV and proteome-analyzed by LC-MS/MS. (B) Significantly increased (red; >1.5-fold up) or decreased (blue; >1.5-fold down) proteins (FDR < 5%) in the different subcellular compartments (see Table 1 for details on clusters). (C) Volcano plot of the proteins detected upon UV treatment, including significantly (FDR < 5%) increased (red) and decreased (blue) proteins. (D) GO categories of human (orange) and C. elegans (blue) annotated proteins.

Figure 2

Proteostatic Shift Leads to Induction of Autophagy, which Is Required for UV Resistance (A) Proteostatic shift from protein synthesis and degradation mechanisms toward autophagy. (B) Immunoblotting of the autophagy marker LGG-1(I) and LGG-1(II)::GFP. LGG-1 becomes lipidated after UV-induced DNA damage and starvation (representative of three independent experiment shown). (C) WT, atg-3(bp412), and atg-9(bp564) L1 larvae were irradiated or mock treated, and developmental stages were evaluated 48 hr later. An average of three independent experiments per strain and dose is shown; >15 individuals were analyzed per experiment. Error bars denote standard deviation. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (two-tailed t test compared with WT).

Figure 3

Response to Persistent DNA Damage Correlates with Starvation Stress and Aging Proteomes (A–C) Correlation analyses (A) between proteome of xpc-1;csb-1 double mutants (FDR < 5%) and transcriptome of xpa-1 mutants after UV treatment (similarly regulated proteins and genes in red and green; specific protein clusters are detailed in Table S3), (B) between proteins detected in xpc-1;csb-1 double mutants upon UV treatment versus aging in WT worms (p < 2.2 × 10⁻¹⁶ for the three Pearson correlation coefficients, r), and (C) between proteins changed in abundance of at least 2-fold (FDR < 5%) in xpc-1;csb-1 double mutants upon UV treatment versus starvation.

Figure 4

Attentuated FA Synthesis and Alterations in Lipid Classes (A) Key members of the FA biosynthetic coupled to SL and phospholipid metabolic pathways were significantly decreased in abundance in xpc-1;csb-1 double mutants upon starvation and UV treatment. (B) TLC detection and quantification (histogram) of FAs and triacylglycerols in xpc-1;csb-1 double mutants upon UV and starvation. (C and D) Changes in the amount of three SLs subclasses (ceramides, sphingomyelins, and glucosylceramides) (C) and of the five glycerophospholipids subclasses (phosphatidylcholine [PC], phosphatidylethanolamine [PE], PI, phosphatidylserine [PS], and phosphatidylglycerol [PG]) (D) assessed by MS analysis. Significant levels of pairwise comparisons are indicated by p values: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

Network of Interactions between Proteins that Are Significantly Regulated in xpc-1;csb-1 Double Mutants upon UV Treatment FDR < 5%. Dark blue, >1.5-fold downregulated; dark red, >1.5-fold upregulated. Different shapes indicate the significantly changed phosphosites normalized to the proteome.

Figure 6

Network Analysis of Proteome and Phosphoproteome Alterations in Response to Persistent DNA Damage (A) Network of interactions between proteins regulated in xpc-1;csb-1 double mutants upon UV treatment. Symbols are as follows: full circles, proteins detected by MS as downregulated (blue) or upregulated (red) or not significantly regulated (white); dotted circles, proteins that are not quantified by MS; and stars, phosphopeptides detected by MS as decreased (blue) or increased (red). p < 0.05. (B) WT, aak-2(gt33), and aak-2(ok524) L1 larvae were irradiated or mock treated, and developmental stages were evaluated 48 hr later. An average of three independent experiments per strain and dose is shown; >540 individuals were analyzed per experiment. Error bars show standard deviation; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗ p < 0.001 (two-tailed t test compared with WT).