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

Katarzyna D. Arczewska 1 2 Gisele G. Tomazella 2 Jessica M. Lindvall 0 Henok Kassahun 2 Silvia Maglioni 5 6 Alessandro Torgovnick 5 6 Johan Henriksson 4 Olli Matilainen 3 Bryce J. Marquis 7 Bryant C. Nelson 7 Pawel Jaruga 7 Eshrat Babaie 2 Carina I. Holmberg 3 Thomas R. Bu rglin 4 Natascia Ventura 5 6 Bernd Thiede 2 Hilde Nilsen 2 0 Huddinge Genomics Core Facilities, Karolinska Institutet, Department of Biosciences and Nutrition , SE 141 57 Huddinge, Sweden 1 Biochemistry and Molecular Biology Department, The Centre of Postgraduate Medical Education , Marymoncka 99, 01 813 Warsaw Poland 2 The Biotechnology Centre, University of Oslo , P.O. Box 1125 Blindern, 0317 Oslo, Norway 3 Research Programs Unit, Molecular Cancer Biology Program, and Institute of Biomedicine , Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8) FI-00014 University of Helsinki Finland 4 Department of Biosciences and Nutrition & Center for Biosciences, Karolinska Institutet , Halsovagen 7, Novum, SE 141 83 Huddinge, Sweden 5 Institute of Clinical Chemistry and Laboratory Medicine of the Heinrich Heine University, and the IUF - Leibniz Research Institute for Environmental Medicine , Auf'm Hennekamp 5040225 Duesseldorf, Germany 6 Laboratory of Signal Transduction, Department of Biomedicine and Prevention, University of Rome ''Tor Vergata'' , Italy 7 National Institute of Standards and Technology (NIST), Materials Measurement Laboratory , 100 Bureau Drive, M/S 8300 Gaithersburg, MD 20899-8300 USA - Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and downregulation 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 transcriptionblocking 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 DDB1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant. Stochastic accumulation of oxidative DNA damage has been regarded as a major contributor to age-related functional loss ever since Harman formulated the original hypothesis of the oxidative damage theory of aging (1). A logical extension of this theory is that DNA repair processes should contribute to increased life expectancies. The existence of accelerated aging syndromes associated with DNA repair defects supports this model (2). Systematic gene expression profiling of segmental progeroid Nucleotide Excision Repair (NER)-defective mice has demonstrated that suppression of insulin-like signaling (ILS) pathways and activation of oxidative stress response pathways are associated with segmental progeroid phenotypes (1,36). Suppression of ILS per se is associated with lifespan extension (7,8). The transcriptomic modulation in segmental progeroid NERmutants is therefore believed to reflect a survival response to accumulation of transcription-blocking oxidative DNA damage (9,10), but important questions remain to be answered. Firstly, given that Base Excision Repair (BER) is more important than NER in repairing oxidative DNA damage, it is puzzling that similar accelerated aging syndromes are not seen in BER-deficient mouse models (11). Secondly, we do not know which types of lesions are responsible for age-related functional loss, although the fact that NER-but not BER mutants-show the more severe phenotypes would point to a role for 8,50cyclopurines as these are the only oxidized bases known to be a substrates for NER but not BER (12). Thirdly, there is little direct evidence to suggest whether passive accumulation of DNA damage is sufficient to cause these phenotypes or whether it is an active process that can be modulated genetically. Caenorhabditis elegans (C. elegans) is frequently used to study genetic factors influencing longevity (13). C. elegans is also well suited to reveal phenotypes that may be masked in mammals due to extensive redundancy of BER enzymes since NTH-1 is the only known DNA glycosylase dedicated to removing oxidized bases in this animal (14). Moreover, the NER pathway is highly conserved (15), with global genome repair (GG-NER) primarily protecting germ cells and early embryos whereas transcription-coupled repair (TC-NER) becomes more important in later developmental stages (16). In mammalian cells GG-NER depends on UV-DDB and XPC/hHR23 for DNA-damage detection whereas TC-NER is initiated by stalling of RNA polymerase II on a lesion and depends on CSB (17). Both branches depend on XPA for damage verification and formation of the preincision complex (18). C. elegans xpa-1 mutants are UV-sensitive and have reduced capacity to repair UV-induced DNA damage (see (15) for a recent review). Contradictory reports exist as to whether NERdeficient xpa-1 mutant animals have shortened lifespan (discussed in (15)). We previously showed a small, but significant reduction of median lifespan in xpa-1 mutants that was accompanied with up-regulation of oxidative stress response genes (19). Moreover, we showed that deletion of the BER enzyme NTH-1 reversed the transcriptome changes and restored normal lifespan of the xpa-1 mutants (19) supporting a model where the NTH-1 enzyme itself generates a response that results in lifespan shortening in xpa-1 mutants. Here, we provide evidence for an active reprogramming response in xpa-1 mutants. MATERIALS AND METHODS For more detailed experimental procedures please refer to Supplementary Materials and Methods. Caenorhabditis elegans and bacterial strains C. elegans strains were cultured at 20 C on solid Nematode Growth Medium (NGM) agar plates using standard procedures (20). Wild type (WT) Bristol N2, RB877 nth-1(ok724) III, RB864 xpa-1(ok698) I and CL2166 [dvIs19[pAF15(gst-4::gfp::NLS)] III] (21) C. elegans, as well as Escherichia coli HT115(DE3) and OP50 were obtained from the Caenorhabditis Genetics Centre (CGC) (University of Minnesota, St. Paul, MN, USA), funded (...truncated)