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Gene Expression Responses to DNA Damage Are Altered in Human Aging and in Werner Syndrome

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Gene Expression Responses to DNA Damage Are Altered in Human Aging and in Werner Syndrome Oncogene (2005) 24, 5026–5042 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Gene expression responses to DNA damage are altered in human aging and in Werner Syndrome Kasper J Kyng1,2, Alfred May1, Tinna Stevnsner2, Kevin G Becker3, Steen Klvra˚ 4 and Vilhelm A Bohr*,1 1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA; 2Danish Center for Molecular Gerontology, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark; 3Gene Expression and Genomics Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA; 4Institute for Human Genetics, University of Aarhus, Denmark The accumulation of DNA damage and mutations is syndromes, caused by heritable mutations inactivating considered a major cause of cancer and aging. While it is proteins that sense or repair DNA damage, which known that DNA damage can affect changes in gene accelerate some but not all signs of normal aging (Hasty expression, transcriptional regulation after DNA damage et al., 2003). Age is associated withan increase in is poorly understood. We characterized the expression of susceptibility to various forms of stress, and sporadic 6912 genes in human primary fibroblasts after exposure to reports suggest that an age-related decrease in DNA three different kinds of cellular stress that introduces repair may increase the susceptibility of cells to agents DNA damage: 4-nitroquinoline-1-oxide (4NQO), c-irra- causing DNA damage. Reduced base excision repair has diation, or UV-irradiation. Each type of stress elicited been demonstrated in nuclear extracts from aged human damage specific gene expression changes of up to 10-fold. skin (Xu et al., 2000), and inefficient repair withage A total of 85 genes had similar changes in expression of after nitroquinoline-N-oxide was found in mice (Zahn 3–40-fold after all three kinds of stress. We examined et al., 2000) and UV repair has been shown to decrease transcription in cells from young and old individuals and at high age in peripheral lymphocytes (Lambert et al., from patients with Werner syndrome (WS), a segmental 1979). DNA damage accumulation withage does not in progeroid condition with a high incidence of cancer, and itself suggest decreased repair capacity, and other found various age-associated transcriptional changes experiments showed no age-associated change in DNA depending upon the type of cellular stress. Compared to repair (Collier et al., 1982). Since repair is not perfect, young individuals, both WS and old individuals had mutations in DNA are expected to accumulate over time similarly aberrant transcriptional responses to c- and even withnormal repair capacity. One study demon- UV-irradiation, suggesting a role for Werner protein in strated an age-associated shift in the pattern of DNA stress-induced gene expression. Our results suggest that repair: in old age, strand break repair mechanisms aberrant DNA damage-induced gene regulation may became more important, while replication repair was contribute to the aging process and the premature aging reduced (Niedermuller et al., 1985). This was interpreted in WS. as a consequence of altered genetic expression during the Oncogene (2005) 24, 5026–5042. doi:10.1038/sj.onc.1208692; aging process. Recently, Seluanov et al. (2004) studied published online 16 May 2005 the ability of young, presenescent, and senescent normal human fibroblasts to repair double-strand breaks in Keywords: microarray; Werner syndrome; DNA transfected DNA by using a fluorescent reporter damage; aging substrate. They found that DNA end joining becomes less efficient and more error-prone during cellular senescence and proposed this as a likely mechanism underlying the age-related genomic instability and Introduction higher incidence of cancer in the elderly. It has been suggested that aging is characterized by a DNA damage induced by cellular metabolism and distinct reprogramming of gene expression (Murano environmental stress is thought to contribute to aging et al., 1991; Lee et al., 1999, 2000; Ly et al., 2000; Zou and the accompanying deterioration of organ function, et al., 2000; Kyng et al., 2001, 2003b; Weindruch et al., slowing of metabolism, and increased carcinogenesis. 2001; Welle et al., 2001), but little is known of A role of genome instability in the aging process is transcriptional regulation after DNA damage in aged supported by the existence of segmental progeroid individuals. Descriptions of coordination of gene expression in response to diverse pathogenic microbes *Correspondence: VA Bohr; E-mail: [email protected] have revealed valuable information on how immune Received 15 December 2004; revised 8 March2005; accepted 8 March cells respond to stress (Huang et al., 2001). In yeast, 2005; published online 16 May 2005 the stress response involves both damage-specific Transcription after DNA damage in aging and WS KJ Kyng et al 5027 transcriptional changes and a common set of genes tion by multiple repair systems, and 4NQO is unique referred to as the environmental stress response (ESR), because WS cells have a known sensitivity to this agent. which are regulated in response to several types of stress (reviewed in Gaschand Werner-Washburne,2002). Sucha coordination of gene expression in response to Results multiple DNA-damaging agents has not been reported in humans, and the pattern of stress-specific and Overview common pathways in human aging is not understood. It is possible that specific shortcomings in stress-induced To investigate age-associated changes in stress-induced gene expression are essential to the development of the gene regulation, we analysed data from approximately aging phenotype. This notion prompted us to explore 7000 human genes. Genes were characterized as stress age-related transcription changes after stress, thus responsive if they changed their expression by threefold mimicking the challenges faced during in vivo aging. or more in cells from young donors (n ¼ 6) in at least one Werner syndrome (WS) is a human segmental time point upon exposure to 4NQO, g-irradiation, or progeria syndrome withmany features of thenormal UV-irradiation. Of 2868 genes withgene ontology t aging process. WS cells display various forms of (GO) annotation, 449 (15.7%) were included in the genomic instability and while they are not hypersensitive further analysis (see Supplementary Table 1). Many to most DNA-damaging agents including UV irradia- more genes changed significantly but failed to make the tion (Poot et al., 2001), they are particularly sensitive to threefold cutoff, which is very restrictive relative to the carcinogen 4-nitroquinoline-1-oxide (4NQO). The comparable studies (Ly et al., 2000; Nantel et al., 2002; mechanisms underlying this particular hypersensitivity Lu et al., 2004). Thus, working from this set of genes are not understood, but are widely considered as an should be considered a conservative representation of important feature of WS at the cellular level. the actual changes. Figure 1 presents a graphical A recent review underlines the value of accumulated overview of stress-induced transcriptional reprogram- gene expression data in the understanding of complex ming in young-, old-, and WS donors. Analysis of the biological processes (Smithand Greenfield, 2003). This transcriptional response to individual DNA-damaging would be particularly relevant in the aging process, agents in young donors revealed that a common set of where several different mechanisms have been thought 85 genes was regulated after all of the stress types tested to be causative, and thus it would be valuable to use (Figure 2). For consistency withtheterminology global genome expression as a mirror reflecting the established in yeast, we refer to these genes as the diversity of cellular processes affected. human environmental stress response (H-ESR). In We have previously characterized gene expression addition, eachDNA-damaging agent was able to differences in untreated primary fibroblasts (Kyng et al., exclusively alter the expression of specific subsets of 2003b) and found that the steady-state transcription genes (Figure 2). Table 1 shows the number of stress patterns in old age and WS were very similar. In responsive genes in cell lines from aged and WS donors accordance with this finding it has been suggested that relative to young donors. We observed that the response several phenotyical aspects WS are secondary conse- in WS was particularly defective after 4NQO, and rather quences of aberrant gene expression (Nakura et al., proficient after UV. To further explore the age- 2000). Gene expression is changed to adapt to cellular associated decline in stress response capacity, we stress (Lee et al., 1995; Moradas-Ferreira and Costa, proceeded to investigate the degree to which different 2000; Volkert and Landini, 2001; Kyng et al., 2003a; pathways were affected. Moggs and Orphanides, 2003), and this adaptation is likely to be affected in aging because of constant Common stress response genes exposure to cellular stress. Here, we have explored how gene expression is Common stress response genes are listed in Table 2. In coordinated in response to multiple types of DNA young donors 59 of the common genes were induced and damage, and how specific stress response pathways are 29 repressed with an overlap of three genes that were aberrant in aging and WS. We were particularly induced or repressed depending on the stress type. The interested in resolving whether transcription patterns direction of change for each gene was generally the same reflect the relative sensitivities to DNA-damaging agents in young, old, and WS donors (Table 2). The common in WS and old donors. To answer these questions, we stress response (H-ESR) comprised 3% of 2868 GO- used cDNA microarrays to study expression changes of annotated genes analysed. In old and WS, a large part of 6912 RNA polymerase II transcribed genes, generating the H-ESR genes were only regulated after certain types approximately one million data points.
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