ARTICLES Persistent transcription-blocking DNA lesions trigger somatic growth attenuation associated with longevity George A. Garinis1,2, Lieneke M. Uittenboogaard1, Heike Stachelscheid3,4, Maria Fousteri5, Wilfred van Ijcken6, Timo M. Breit7, Harry van Steeg8, Leon H. F. Mullenders5, Gijsbertus T. J. van der Horst1, Jens C. Brüning4,9, Carien M. Niessen3,9,10, Jan H. J. Hoeijmakers1 and Björn Schumacher1,9,11 The accumulation of stochastic DNA damage throughout an organism’s lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative stress. Our findings suggest that the accumulation of DNA damage in transcribed genes in most if not all tissues contributes to the ageing-associated shift from growth to somatic maintenance that triggers stress resistance and is thought to promote longevity. Ageing represents the progressive functional decline that is exempted levels as a result of pituitary dysfunction (Snell and Ames mice) — have an from evolutionary selection because it largely occurs after reproduc- extended lifespan17–20. Thus, there are two principal components of ageing: tion and beyond the lifespan normally reached in natural habitats1,2. the stochastic accumulation of damage, and genetic pathways that regulate Accumulation of DNA damage is considered not only a key cause of longevity. However, it is unknown how stochastic damage events are linked cancer but also a driving force of ageing3–6. DNA damage invariably to genes that regulate longevity. accumulates during the lifespan of organisms despite sophisticated DNA The effect of DNA damage on organismal ageing becomes appar- repair systems7–9. In accordance with random damage accumulation, a ent when DNA damage accumulates rapidly early in life as a result of clonal population of Caenorhabditis elegans shows chance variation in defects in genome maintenance, leading to segmental progeroid (pre- lifespan distribution under identical environmental conditions, suggest- mature ageing-like) conditions3,6,21–23. Prime examples are nucleotide ing that ageing may have a strong stochastic component10,11. excision repair (NER) defects6. NER removes a wide range of helix- In contrast to models of stochastic damage as an underlying cause of age- distorting lesions, including UV-induced damage, either by global ing, longevity is also regulated genetically12. Some closely related species, for genome (GG)-NER that repairs lesions throughout the genome or by instance, show great diversity of lifespan, indicating that genetic alterations transcription-coupled repair (TCR), which removes damage in actively can determine longevity13. Single gene mutations in the IGF-1 pathway transcribed genes. Defects in GG-NER lead to skin-cancer-prone xero- extend lifespan in C. elegans and Drosophila14,15. In mammals, GH-mediated derma pigmentosum (XP), whereas defects in TCR lead to the progeroid GH receptor (GHR) signalling in various cell types induces the secretion conditions Cockayne syndrome (CS), trichothiodystrophy (TTD) and of IGF-1, which regulates somatic growth through the activation of IGF-1 XPF-ERCC1 progeria (XFE), which is additionally linked to interstrand receptor (IGF-1R) signalling; together these comprise the somatotropic crosslink (ICL) repair21,24. Mouse mutants with engineered mutations in axis16. Mice with decreased IGF-1 signalling — whether through IGF-1R both Csb and Xpa or in Ercc1 mimic the repair deficiencies of patients heterozygosity, Ghr knockout, overexpression of Klotho, or decreased GH with CS and those with XFE, respectively. Recently, we showed that 1MGC Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands. 2Institute of Molecular Biology and Biotechnology, FORTH, GR70013 Heraklion, Greece. 3Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany. 4Institute for Genetics, University of Cologne, 50674 Cologne, Germany. 5Department of Toxicogenetics, LUMC, 2300RC Leiden, The Netherlands. 6Erasmus Center for Biomics, Erasmus Medical Center, 3000DR Rotterdam, The Netherlands. 7Integrative Bioinformatics Unit, Institute for Informatics, Faculty of Science, University of Amsterdam, 1098SM Amsterdam, The Netherlands. 8National Institute of Public Health and the Environment (RIVM), Laboratory of Toxicology, Pathology and Genetics (TOX), 3720BA Bilthoven, The Netherlands. 9Cologne Excellence Cluster for Cellular Stress Responses in Aging Associated Diseases (CECAD), 50674 Cologne, Germany. 10Department of Dermatology, University of Cologne, 50931 Cologne, Germany. 11Correspondence should be addressed to B.S. (e-mail: [email protected]) Received 17 October 2008; accepted 5 February 2009; published online 12 April 2009; DOI:10.1038/ncb1866 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2009 Macmillan Publishers Limited. All rights reserved. ARTICLES a 10 9 genes involved in IGF-1/GH signalling21,25, which is normally associated 8 26 7 with lifespan extension . It has been hypothesized that somatotropic P 6 attenuation becomes progressively installed with advancing age as a 5 )+( )-( –log 4 result of gradually accumulating damage, and delays the onset of age- 3 related pathology and extends lifespan21,25. It is unknown how suppres- 2 1 sion of the somatotropic axis is triggered and whether somatic growth 0 attenuation is a direct response to specific DNA damage or an indirect consequence of damage-driven tissue degeneration. Here we present DNA repair evidence that the accumulation of transcription-blocking lesions is a Angiogenesis Ubiquitin cycle Mitotic cell cycle DNA metabolism Lipid metabolism Immune response primary cause for attenuation of the somatotropic axis with age and thus Response to stress Signal transduction Protein metabolism Protein metabolism Cell cycle regulation Antigen presentation Response to wounding Regulation of apoptosis links DNA damage to genes that regulate longevity. DNA replication initiation Regulation of metabolism Cellular defence response Response to DNA damage Macromolecule metabolism Amino-acid phosphorylation Intracellular signalling cascade Amino-acid dephosphorylation Growth factor receptor signalling Steroid hormone receptor activity Response to endogenous stimulus RESULTS b Birc5 Apbb2 Ckap2 Prkce DNA damage impinges on pathways associated with longevity Lrdd Erbb2ip Amid Bmpr2 regulation Bnip3l Hbegf Traf4 Plcb4 Moap1 Fyn Inherited defects in NER provide clear-cut links with cancer as well as Topors Snx24 6 Apoptosis Trp53inp1 Morf4l1 premature ageing . We therefore examined whether DNA damage by Pea15 Cova1 Tnfaip3 Ghr Ankrd17 Igf1r ultraviolet (UV) irradiation induces gene-expression responses that nor- Brca1 Rasa2 Chek1 Zfyve9 mally occur in old age. To this end, primary mouse dermal fibroblasts Clspn Pld1 Rad51ap1 Ywhae m/m Fen1 Grb14 (MDFs) from selected NER mouse mutants with severe progeria (Csb / Uhrf1 Gulp1 −/− m/m −/− Rad54l Rasgrp3 Xpa ), mild progeria (Csb ) or no progeria (Xpa ) and wild-type lit- Cdkn1a Socs2 Pcna Somatotropic axis and growth Arhgef12 Mpg Ube2e3 termates at 15 days of age were exposed at early passage (less than six) to Xrcc2 Igfbp4 −2 −2 Csb Rasgrf1 very low (0.6 J m ) and to moderate (4 J m ) UV doses. The sensitivity of Supt16h Kif16b Ptpn11 Ralgps1 −2 Chaf1b Ebpl NER-deficient cells exposed to 0.6 J m UV is equivalent to that of wild- Exo1 Hibadh −2 Rfc5 AF397014 type cells after 4 J m UV (Supplementary Information, Fig. S1). These Topors Mapk14 Response to DNA damage Rad1 Ak5 Rad23a Akap9 UV doses give rise to a maximum of one lesion in 300 or 50 kilobases at Trpc2 Atp11c −2 27 Cxcl1 Hs2st1 0.6 or 4 J m , respectively . Consistent with the presence of randomly Arl6ip2 Arsj Chst4 Atrnl1 Cxcl12 AI317237 distributed lesions in only a small fraction of genes, we did not detect a H2-DMa Cyb5r4 Cmtm7 Maoa Il10rb global transcriptional repression 6 h after UV treatment, because equal Ncf1 Ppa2 Gdf10 Pik3ca numbers of genes were upregulated and downregulated (Supplementary Tap1 Sptlc2 Fcgr1 Gpd2 Nfat5 Oxidative metabolism Pcca Information, Tables S1–S5). A large fraction of UV-induced genes con- Mical2 Immune reponse Lefty1 Tlr4 Dip2c tained relatively long open reading frames, suggesting that the changes Aldh1a3 Tpk1 Nod 4631427C1 Agps Pitpnc1 in expression represent a trans response rather than a cis effect of tran- Lass6 Hecw2 Hmgcs1 C3HC4-1 scriptional blockage. To validate the microarray results, we quantitatively Hmgcs1 Cul3 Plcb4 Tbl1xr1 Tmem23 Cblb confirmed statistically significant changes in gene expression greater than Soat1 Itch Pld1 Phr1 ±1.2-fold by quantitative real-time polymerase chain reaction (qPCR) Lass6 Kcmf1 Ppap2b Znrf3