Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1

Abstract

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Figure 1.

Comparative quantitative proteomics by IPTL. (A) Experimental design. C. elegans N2 and xpa-1 Lys-C resulting peptides were crosswise labeled using the IPTL technique, and combined 1:1 before submission to LC-MS/MS. As an example, N-terminal light/C-terminal heavy (red) and N-terminal heavy/C-terminal light (blue) pairs (10 ratio counts input) from the mass spectrum of a selected peptide identified as MVTIVDPHIK (inset with the detected y and b series ions represented), present in the protein F40F9.6, 2.3-fold up-regulated. (B) Quantitative analysis of DHS-21 and STI-1 protein levels by western blotting. Protein levels were normalized to alpha actin and presented as average fold changes ± SD from four independent replicates.

Figure 2.

GSEA reveals over-represented BPs regulated in xpa-1 mutants. Differential gene products from the proteomic (A) and transcriptomic (B) analysis were imported into Cytoscape and analysed using the BiNGO plug-in for BPs enrichment analysis. Up- and down-regulated genes are highlighted in blue and red, respectively. The nodes are colored yellow to orange, representing increasing statistical significance after Benjamini–Hochsberg correction (from P < 0.05 to P < 7 * 10⁻⁷, respectively).

Figure 3.

Bioenergetic imbalance and induction of antioxidant defense genes in xpa-1. (A) Transcript levels of sod-3, gst-4 and hsp-1 were measured by qRT-PCR. All transcript levels were normalized to that of gamma-tubulin (tbg-1) and presented as average ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01; Student’s t-test. (B) Pgst-4::gfp transgene expression was measured by western blotting in CL2166 fed empty vector control or xpa-1 RNAi. Protein levels were normalized to alpha actin and presented as fold-changes relative to control as average ± SEM from two independent biological replicates. Steady-state ROS levels were measured using the ROS sensitive probe H2-DCF-DA and are presented as fold-change of relative fluorescence units (RFUs) normalized to WT as average ± SEM from three independent biological replicates. ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II. The ATP levels are presented as fold-change of relative luminescence units (RLUs) normalized to the WT and given as average ± SEM from three independent replicates. (C) Polyubiquitinated proteins accumulate in xpa-1. Three separate nematode lysates were separated by 7.5% SDS-PAGE and blotted against polyubiquitinated proteins or alpha-tubulin as a loading control. Note that the exposure times of the film vary between the three independent experiments.

Figure 4.

Reprogramming serves as a survival response in xpa-1. (A) Body length of WT and xpa-1 nematodes fed with empty vector control (con), sti-1, spc-1 and sma-1 RNAi measured when WT nematodes on control food reached the L4/adult stage. Results are presented as medians (line) or means (square) inside the boxes showing the 25–75 percentile range with whiskers presenting SD. The lengths of 150 (spc-1 and sma-1 RNAi) or 90 (control and sti-1) were measured for each condition. (B) The measured levels of oxidized DNA bases in DNA isolated from WT and xpa-1 mutant nematodes. The DNA lesions were measured using GC/MS/MS and the results represent average lesion levels ± standard deviation from 5 to 7 independent biological replicates. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post-test. *P < 0.05, **P < 0.01. (C) The measured levels of oxidized DNA nucleosides in DNA isolated from WT and xpa-1 mutant nematodes. The DNA lesions were measured using LC/MS/MS and the results represent average lesion levels ± standard deviation from 5 to 7 independent biological replicates. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post-test. *P < 0.05, **P < 0.01.

Figure 5.

NTH-1 and XPC-1 are upstream factors required for transcriptome modulation in xpa-1. (A) Transcript levels of sod-3, aqp-1 and gst-4 were measured by qRT-PCR in WT or xpa-1 nematodes fed with RNAi constructs targeting nth-1, xpc-1, rad-23, ddb-1, csb-1, skn-1 and daf-16. All transcript levels were normalized to that of gamma-tubulin (tbg-1) and presented as fold-changes relative to the WT. (B) Transcript levels measured in WT or xpa-1 mutants in the absence (-) or presence of the antioxidant NAC. (C) Cartoon showing the working model: in the WT situation (top), transcription-blocking lesions (small circle) do not accumulate and are not processed by BER. In the absence of xpa-1 (middle) the NER pathway is not functioning, leaving NTH-1 to bind unprocessed DNA lesions. This generates genomic stress signals and activates reprogramming, which induces ROS generation. When both xpa-1 and nth-1 are missing (bottom) there is no DNA repair enzyme that may bind the lesion site, the lesion is bypassed and no reprogramming is initiated. The results are presented as average ± SEM from at least three independent replicates. *P < 0.05, **P < 0.01; Student’s t-test.