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.

Active transcriptomic and proteomic reprogramming in theC. elegansnucleotide excision repair mutant xpa-1

“…As expected from the near normal phenotype, xpa-1 mutants do not show the extensive transcriptomic modulation seen in segmental progeroid NER-defective mice. However, we, 31,36 and others, 51 identified a small yet informative set of genes differentially expressed in xpa-1 mutants. Gene set enrichment analyses (GSEA) revealed that the biological process (BP) "determination of adult lifespan" was the only overrepresented BP among the regulated genes.…”

“…Using this strain we could show that NER indeed contributes to repair of oxidative DNA damage in C. elegans as the steady-state levels of the oxidized DNA bases formamidopyrimidines (FapyGua and FapyAde) and 8-hydroxyadenine (8-OH Ade), which are classical BER substrates, are significantly increased in xpa-1 (ok698) mutants. 36 Contradictory reports exist as to whether NER-deficient xpa-1 mutants have shortened lifespan (discussed in ref. 35).…”

“…31 This result was independently confirmed in a collaborating laboratory which also showed that normal median lifespan was regained if we grow the same strain on E. coli HT115(DE3). 36 Hence, the lifespan of xpa-1 mutants is somewhat shorter than the isogenic wild-types, but it is influenced by external factors such as nutrition. Phenotypic variation induced by diet is well known in C. elegans and impinges on many of the same processes that are regulated in DNA repair mutants.…”

“…Overexpression and nuclear translocation of the SKN-1 target gene GST-4 confirmed that xpa-1 mutants experience oxidative stress. 31,36 We also used quantitative proteomics to assess whether the transcriptomic signatures were reflected at the level of the proteome. We used a newly developed technique called isobaric peptide terminal labeling (IPTL) technology, 52 which can be used to label proteins in freshly prepared extracts from whole worms 36 or from isolated tissues like the germline.…”

“…31,36 We also used quantitative proteomics to assess whether the transcriptomic signatures were reflected at the level of the proteome. We used a newly developed technique called isobaric peptide terminal labeling (IPTL) technology, 52 which can be used to label proteins in freshly prepared extracts from whole worms 36 or from isolated tissues like the germline. 53 It is a very good alternative for C. elegans proteomic studies where metabolic labeling has some limitations.…”