DNA, the central molecule of aging

Accepted Manuscript Title: DNA, the central molecule of aging Author: Peter Lenart Lumir Krejci PII: DOI: Reference:

S0027-5107(16)30007-0 http://dx.doi.org/doi:10.1016/j.mrfmmm.2016.01.007 MUT 11531

To appear in:

Mutation Research

Received date: Revised date: Accepted date:

17-12-2015 16-1-2016 30-1-2016

Please cite this article as: Peter Lenart, Lumir Krejci, DNA, the central molecule of aging, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis http://dx.doi.org/10.1016/j.mrfmmm.2016.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: DNA, the Central Molecule of Aging Peter Lenart1 and Lumir Krejci*1,2,3

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Department of Biology, Masaryk University, Brno, Czech Republic; 2International Clinical Research Center, Center for Biomolecular and Cellular Engineering, St. Anne’s University Hospital Brno, Brno, Czech Republic; 3National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Abstract: Understanding the molecular mechanism of aging could have enormous medical implications. Despite a century of research, however, there is no universally accepted theory regarding the molecular basis of aging. On the other hand, there is plentiful evidence suggesting that DNA constitutes the central molecule in this process. Here, we review the roles of chromatin structure, DNA damage, and shortening of telomeres in aging and propose a hypothesis for how their interplay leads to aging phenotypes. Keywords: DNA; aging; chromatin structure, telomeres; DNA damage, DNA repair; mutagenesis 1. INTRODUCTION Aging is a complex biological process resulting in the decline of almost all physiological functions, which in turn leads to a time-dependent increase in mortality. Many theories have tried to explain the aging process, but none is universally accepted [1, 2]. Although many biomolecules could play a role in aging, DNA seems to be the most relevant. This is supported by several arguments. First, syndromes of accelerated aging are often associated with defects in DNA repair genes [3, 4]. Second, changes of chromatin structure, shortening of telomeres and accumulation of DNA damage are all associated with aging and life span [5-9]. These chromosomal changes do not act in isolation but are rather tightly interconnected. Changes of chromatin structure can accelerate shortening of telomeres [10] alter susceptibility to DNA damage [11] and modify transcription [12] thus influencing almost all cellular functions. Vice versa, DNA damage can lead to changes of chromatin structure [13] and accelerate telomere shortening [14]. Here we review how these three types of chromosomal changes and their interplay influence the aging process. 1.1. Chromatin Structure Chromatin is a nucleoprotein complex that can be understood as a dynamic, three-dimensional, higherorder structural state of a chromosome. The basic unit of chromatin is a nucleosome consisting of four pairs of core histones (H2A, H2B, H3, H4) around which 147 bases of DNA are wrapped [15]. Higherorder chromatin structure is also formed by linker H1 histone and other non-histone proteins. There exists two basic types of chromatin: highly condensed heterochromatin or loosly condensed euchromatin, characterized by typically transcriptional inactivity and resistance to DNA damage or transcriptional activity and susceptibility to DNA damage, respectively [11]. *Address correspondence to this author at the Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5/A7, Brno 62500, Czech Republic, Telephone: +420-549-493-767; Fax: +420-549-492-556; E-mail: [email protected] Therefore, it is not surprising that several studies have shown depletion of core histones to be associated with aging. In yeast, histone concentration decreases with age and seems to be directly related to aging, since their overexpression leads to a 65 % increase in replicative life span [8]. The possible mechanism might be changes in transcription, as loss of nucleosome in yeast was reported to cause globally increased gene expression [16]. In addition, decreases in H3 and H4 histone levels also have been observed in human senescent fibroblasts, where their concentrations were reported as reduced by half compared to

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levels in young cells [5, 17]. Although synthesis of new histones diminishes with age, it is notable that their expression actually increases [18]. Even as cells are trying to stabilize histone levels, therefore, this might be counteracted by an increased rate of mRNA degradation or increasingly ineffective translation. It would therefore be interesting to test whether overexpression of histones would extend the life span of human cells. Chromatin structure can be altered not only by changes in the number of histones but also by their posttranslational modifications. More than 60 modification sites have been identified on histones, which, together with the 8 basic types of modifications, means the number of specific modifications (type and position) is immense. Histone modifications are known to play important roles in regulation of many cellular processes such as replication, transcription, and DNA repair [19], and so it is not surprising that types and amounts of modifications change with age. For instance, acetylation of lysine 16 on histone 4 (H4K16ac) increases with age in yeast [5, 20]. Other examples include increases in H3K9ac and reductions in H3K56ac [5]. Acetylation in general removes positive charge from lysine and could be expected to weaken the binding of DNA to histone and, in turn, make chromatin structure looser. Indeed, H4K16ac has been implicated in determining chromatin structure and influencing the interaction between non-histone proteins and chromatin [21, 22]. In addition, the dynamic status of the modification plays an important role, as Sir2, the main deacetylase regulating this modification, is also known to extend life span in several invertebrates such as Saccharomyces cerevisiae [23], Caenorhabditis elegans [24], and Drosophila melanogaster [25]. It is not clear, however, if SIRT1, the human Sir2 orthologue [26, 27], can extend life span in mammals, since overexpressing SIRT1 in all mouse tissues was shown to have no lifeextending effects despite its having a positive effect on several pathologies associated with aging [28]. Surprisingly, another study overexpressing SIRT1 in mouse brains reported 11 % elongation of life span [29]. It can therefore be speculated that SIRT1 is beneficial to life span only in certain tissues or only at low concentrations. Strong evidence for a connection of histone acetylation with chromatin maintenance and aging can be seen in the effect of the polyamine known as spermidine on life span and aging. In general, polyamines are associated with cell growth [30], their depletion inhibits apoptosis [31], and they are also implicated in carcinogenesis inasmuch as their concentrations are elevated in cancer cells [32]. In addition, it has been shown that yeast as well as mammalian cells synthesize less polyamines with age. Eisenberg et al. have shown that spermidine supplementation extends life span in yeast, nematodes, flies, mice, and also human cells and that this effect is accompanied by hypoacetylation of H3K9, H3K14 and H3K18. Accordingly, inactivation of acetyltransferases responsible for acetylation of these lysines also extended the life span of yeast and decreased the effect of spermidine treatment [33]. Histones modification by methylation of their lysine or arginine residues plays also important role. In contrast to the histone acetylation, methylation can be associated with either active or repressed transcription, depending on the affected residue [34]. While trimethylation of lysine 4 on histone 3 is associated with active transcription [35], trimethlyation of lysine 27 on the same histone is associated with repressed transcription [34]. Both of these modifications are linked to aging. In human senescent fibroblasts H3K4me3 is enriched and occupies new parts of genome [36]. Spreading of H3K4me3 during aging has bean also observed in mouse hematopoietic stem cells [37]. The most direct evidence of role of H3K4me3 in aging comes from C. elegans, where overexpression of RBR-2, the H3K4me3 demethylase, increase life span, while its knockdown has opposite effect. Furthermore knockdown or mutation of genes encoding H3K4 methyltransferases increases life span [38]. In contrast to H3K4me3 mark, H3K27me3 decreases during aging and knockdown of UTX-1, H3K27me3 demethylase extends life span of C. elegans [39]. However, role of H3K27me3 in aging is not as clear, as experiments in D. melanogaster have shown that mutation in H3K27-specific methyltransferase E(Z) increases life span of flies and reduce amount of H3K27me3 [40]. Other histone modifications are less characterized, but may nevertheless influence aging. For example low levels of ubiquitination of H2B were found to be necessary for yeast cells to attain normal life span possibly trough regulating Sir2 recruitment [41]. DNA methylation is also relevant in determining chromatin structure [42, 43] and regulating gene expression [44]. Changes in methylation are typical for cancer cells, but similar changes have been observed also in senescent cells [45–47]. Aging cells exhibit global hypomethylation and local hypermethylation. While hypomethylation is typical for noncoding parts of the genome, sequences near the promotors of several genes regulating the cell cycle are often hypermethylated [48]. Several authors

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have been able recently to use methylation patterns to successfully predict the age of several different tissues, mostly by analyzing methylation of different CpG sites as an aging clock [49–51]. Furthermore, the methylation patterns of genomic DNA were also shown to predict mortality regardless of current health status, life style, and known genetic predispositions [52], thus suggesting a direct relationship between DNA methylation and aging. A causal role of DNA methylation in aging is also suggested by the ability of methionine restriction – a well-known dietary intervention – to prolong the life span of various model organisms [53–56] and human fibroblast [57]. It is expected that this is due to methionine’s serving as a substrate for methyl-transferases and thus affecting the methylation state [58]. 1.2. Telomeres Shortening Telomeres are nucleoprotein complexes protecting coding sequences from replicative shortening of chromosomes. Telomeric DNA consists of a G-rich repetitive sequence (TTAGGG in mammals) bound by numerous proteins forming a shelterin complex which also blocks these ends from being recognized as DNA double-strand breaks (DSBs) [59]. Telomeres progressively shorten after each cell division, limiting the number of divisions of somatic cells. They can be extended by a special enzyme termed telomerase, which in humans is active mostly in embryonic stem cells [60]. Telomeres and telomerase have long been implicated in the aging process, as introduction of telomerase to cells in vitro is able to postpone senescence and promote immortalization [6]. It was later proven that transient telomerase expression did extend the life span of human cells, but this did not result in their immortalization [61]. This led to the hypothesis that telomere shortening is the primary cause of aging and sparked many studies analyzing the impact of telomere length on human health. The first studies defining the role of telomeres in aging of mammals involved mice with knock-out of the genes necessary for telomerase synthesis (Terc or Tert genes). Surprisingly, first-generation mice from these knock-out lines are healthy with no significant changes in phenotype [62, 63], thus indicating that telomerase is not necessary for life and that a lack of phenotype might be due to residual telomerase activity in the organism. Accordingly, the phenotype should be more visible in future generations. Indeed, from the third generation onward these mice have significantly shorter telomeres, decreased fertility, overall frailty, and atrophy of tissues leading to organ malfunctions [64]. Aging is exacerbated in mice defective for the WRN helicase, mutated in Werner syndrome and responsible for telomere maintenance [65, 66]. This is interesting since Werner syndrome is a well-known progeria in humans showing an interaction with different genome maintenance mechanisms [67]. Telomerase is often expressed in cancer cells, since it can allow unrestricted proliferation, and it is therefore not surprising that classical transgenesis increases the incidence of cancer [68–70]. Nevertheless, overexpression of telomerase in mice genetically modified to be more resistant to cancer by increased expression of tumor-suppressor genes led to slower aging and increase in median life span by 40 % [71]. Additionally, cancer incidence is not increased if the telomerase is transduced into adult mice and transduction of telomerase into 1-year-old mice extended their life by 24 % [72]. While the aforementioned findings clearly demonstrate that telomere shortening and accordingly telomerase activity influence aging, the results of comparative studies show that telomere shortening is almost certainly not the primary cause of aging. For example, a study comparing telomerase activity in different rodents found no correlation between this enzyme’s activity and rodent life span. On the other hand, a significant negative correlation with body weight was observed and suggests a possible cancerprotective strategy of organisms [73]. An identical relationship between telomerase activity and body weight was also observed in a study comparing more than 60 mammalian species [74]. Interestingly, a negative correlation between telomere length and life span was found. On the other hand, telomere length can be used to predict the life span of individual zebra finches [75]. Although this seemingly contradictory data could be used to argue that telomeres are rather downstream effectors of aging, in species that evolved to use their proliferation-limiting potential as a protection mechanism against cancer their telomere shortening speed can determine individual life span. Studies in humans analyzing the relationship between telomere length and health and mortality have led to findings no less interesting. The initial study analyzing 143 subjects older than 60 showed a strong correlation between shorter telomeres in leukocytes from periphery blood and increased mortality [76]. This result was not confirmed, however, by more recent studies involving considerably larger cohorts. While a study on a cohort of 3,075 healthy men and women aged 70–79 found no significant correlation between telomere length and mortality, it did show a correlation between telomere length and years of healthy life span [77]. Short telomeres were also associated with risks of many illnesses such as various types of cancer [78], renal dysfunction [79], autism [80], mortality from cancer in patients with chronic obstructive pulmonary disease [81], and many others. In principle, however, this kind of study is only correlative and cannot prove a causal relationship. The situation is also complicated by the fact that longer telomeres were also associated with risks of deadly diseases. While one study found a correlation between

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long telomeres and a risk of lung cancer in never-smoking women [82], another associated longer telomeres with susceptibility to colorectal carcinoma [83]. Telomere length greater than average was also correlated with a risk of breast cancer [84]. This brief overview illustrates that within the current state of knowledge it remains very difficult or even impossible to objectively evaluate the influence of telomere length on human health. Therefore, a large meta-analysis is needed that could provide more clear evidence. 1.3. DNA Damage As a consequence of endogenous or exogenous influences, the chemical structure of DNA can be altered or lead to breakage of one or both DNA strands. A causal role of DNA damage in aging is strongly suggested by its role in progeria syndromes [3] as well as other indications. One of these is that many lifespan-extending strategies lead to enhanced DNA repair or decreased DNA damage. For example, caloric restriction, the oldest known intervention able to prolong life span, has been reported to decrease agerelated decline of base excision repair (BER) [85], nucleotide excision repair [86], and non-homologous end joining [87]. It also increases expression of SIRT1 [88, 89], which enhances function of homologous recombination [90]. Furthermore the mTOR inhibitor, rapamycin was shown in mice to increase life span [91] and reduce DNA damage and cancer in skin exposed to a DNA damaging agent [92]. DNA adducts are a common type of DNA damage. Formation of DNA adducts is caused mainly by reactive-oxygen species, with 8-oxoguanine (8-oxoG) being commonly used as a marker of oxidative stress [93]. Current data suggest that DNA adducts do not play an important role in aging since even a 20fold increase in 8-oxoG levels in mice did not result in visibly accelerated aging [94]. Another frequent type of DNA damage involves apurinic/apyrimidinic-sites (AP sites), which arise from spontaneous depurination or through repair of modified bases via BER. Creation of AP sites eventually leads to base replacements, frame shift mutations and DNA breaks, and accumulation of AP sites with age is supported by their 7-fold increase in leukocytes from old donors when compared to young donors [95]. In addition, BER is required for yeast to attain a full chronological life span [96], supporting its role in aging. Singlestrand breaks (SSBs) are the most common type of DNA damage, with more than 10,000 lesions arising per cell per day [97]. This important type of DNA damage is probably not connected to aging, however, since studies analyzing SSBs in mice have found no age-dependent derivation in SSB levels [98, 99]. DSBs are the most dangerous type of damage and arguably are also the most associated with aging. Unrepaired or incorrectly repaired DSBs can lead to loss of a part of a chromosome or to gross chromosomal reengagements and genomic instability, which processes are linked with many diseases including those related to advanced age and DSB repair defects can lead to early aging in mice [100]. DSBs increases with age both in vitro and in vivo [101, 7], as does the time within which cells respond to DSBs, thus indicating the gradual loss of DNA repair efficiency [102]. Accordingly, it was recently shown that inducing DSBs in mice livers by expression of the restriction enzyme Sac1 accelerated this organ’s aging [9]. DNA damage response (DDR) is a mechanism by which cells sense damage and trigger corresponding repair. This response includes modification of histones near the damage site. Specifically, H2AX histones are phosphorylated (γ–H2AX) [103–105]. These changes can span as many as even several megabases from the DSBs [106] and as a consequence can also inhibit transcription [107, 108]. However, heterochromatin, which is also induced by aging, limits DDR [109, 110], and this can be attributed to an inability of cells to recognize DNA damage. Such assumption has been challenged, however, since γ– H2AX can be generated successfully even in the very dense structure of mitotic chromosomes [111]. Another possible explanation might be that this reflects changes in chromatin structure rather than recognition of DNA damage itself by DNA repair proteins [112, 113]. A variety of other chromatin modifications also accumulate at sites of DNA damage. Very important is ubiquitination of H2A and H2B that further stimulates cell response to DNA damage [114, 115]. These modifications not only play a role in DNA repair but also influence transcription both locally and globally, and many changes in transcription caused by DNA damage are transitional [116]. In some cases, however, inability to restore a normal chromatin structure after DNA repair can significantly deregulate transcription. One such example is delocalization of the histone deacetylase SIRT1. In response to DNA damage, SIRT1 binds to the damaged site. There, it plays a role in repair, although this leads to its deficiency in other parts of the genome and the depression of SIRT1 regulated genes [13]. This process is similar to changes observed in aging and it seems to be evolutionarily conserved inasmuch as binding of SIR proteins to DNA damage sites also leads to their depression in other parts of the genome and manifestation of an aging phenotype [117].

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Other proteins, too, are implicated in DNA repair and chromatin structure that also regulate life span. Both SIRT1 and yeast Sir2 have important roles in DDR, for example, as they bind to the DNA damage site and there deacetylate several proteins involved in DNA repair and stimulate their activity [118]. DSB repair leads to acetylation of the N-terminal lysines of histones H3 and H4 [117]. This is very intriguing with regard to aging because, as mentioned above, hypoacetylation of the N-terminal lysines of histones is at least partially responsible for the life-extending effects of spermidine [33]. Sirtuins also play an important role in maintaining heterochromatin on repetitive sequences of rDNA and telomeres and prevent aberrant recombination between these repetitive sequences [90, 119]. As would be expected, mice with inactive SIRT6 have increased genomic instability and show accelerated aging [120]. In addition, overexpression of pch-2, which modulates meiotic recombination and helps to maintain ribosomal DNA stability [121] was recently shown to extend the life span of C. elegans and increase resistance to multiple stressors, thereby affecting both DNA and protein integrity. Furthermore, even though the effect of pch-2 modulation is sir-2 independent, the same authors propose a mechanism of pch-2 and sir-2 interaction leading to life extension [122]. In addition, deletion of Exo1, nuclease involved in mismatch repair and recombination, is able to prolong the life span of telomere-dysfunctional mice, possibly by impairing DNA damage signals at DNA breaks [123]. Mutations arise as a consequence of nonfunctional DNA repair or mistakes during replication. Mutations were first mentioned as the primary cause of aging in the middle of the 20th century [124, 125]. It was nevertheless impossible for decades directly to compare mutation rate in differently aged cells. Finally, several studies showed that mutations are indeed accumulating with age and reflect the proliferative capacity of tissues [126–128]. Despite this, small mutations that result from improper repair of base lesions, base mismatches or indels are unlikely to constitute one of the primary causes of aging since transgenic mice with defects in mismatch repair [129] or antioxidant defense [130] exhibit an increase in spontaneous mutation rate, but do not shown any signs of accelerated aging apart from increased cancer incidence. Similarly mice, with defective transcription-coupled repair exhibited early ageing without an increase in mutation frequency [131]. A recent study also confirmed that aging in S. cerevisiae is not caused by an increase in small mutations, since old yeast cells with few or no mutations were dying even though young cells mutated in eight thiol peroxidases, which are enzymes detoxifying hydroperoxides, had twice as many mutations and continued dividing [132]. By contrast, defective repair of DNA DSBs could accelerate aging since mutations in multiple DSB repair genes leads to early aging in mice and people and since chromosomal abnormalities correlate with age [100]. Thus, the accumulation of chromosomal rearrangements might be a good indicator for some age-associated pathologies, such as cancer. Conclusion It is unlikely for such a complex process as aging to be driven just by one specific kind of disturbance. Accordingly, we suggest that aging is caused by the interplay of several types of damage. Even though their relationship may form complex network which is yet to be fully understood, we believe that there is already a traceable pattern suggesting a possible order of their interaction (Fig. 1). We propose that DNA damage serves as trigger of aging. Accumulation of DNA damage, mainly DSBs, leads to chromatin modifications and chromatin’s loosening, which in turn makes chromatin more prone to additional DNA damage. These changes of chromatin structure then affect transcription, and accelerate telomere shortening. Faster telomere shortening may then lead to quicker depletion of stem cells in organisms thus reducing regenerative capacity of tissues. In addition, we suggest that changes in transcription can lead to age-related decrease in physiological functions, since cells with progressively more deregulated transcription can scarcely maintain optimal biological function. Moreover, this notion is strongly supported by fact that changing levels of specific histone modification also influences life span. Finally, unrepaired or incorrectly repaired DNA damage can also lead to mutations, and these can inversely affect all the aforementioned processes. Even though accumulation of small mutations probably does not play a causal role in aging, it can influence any of the individual processes and thus manifest some age-related pathologies, and especially increased incidence of cancer. The accumulation of chromosomal rearrangements could impact aging by directly impairing cell homeostasis or inducing cell senescence. Most importantly, this model indicates not only the possible importance of these processes individually but in particular their combination that eventually leads to aging. It might be this combination that gives to the entire process the complexity that would lead to a determination of direct causality. Nevertheless, it can be expected that future research in this area will shed more light on how changes relating to the DNA molecule affect the aging process. A better understanding of the mechanism will enable us

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to identify appropriate targets for intervention, generate new compounds, and develop new strategies to counteract aging phenotypes. Furthermore, it could be expected that aging research will continue to capture the attention of an ever-increasing number of scientists, since mathematical models clearly demonstrate that to delay aging will have enormous health and economic benefits greatly outweighing the potential benefits of separately addressing heart disease or cancer [133]. Conflict Of Interest The authors declare no conflict of interest. Acknowledgements This work was supported by Czech Science Foundation grants GACR13-26629S and GACR207/12/2323, European Regional Development Fund [Project FNUSA-ICRC, no. CZ.1.05/1.1.00/02.0123] and Research Support Programme [GAMU] - MUNI/M/1894/2014.

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Fig. (1). Interaction between different types of molecular damage and their relationship to aging. DNA damage arises as a consequence of intrinsic or extrinsic influences and when not repaired correctly can lead to mutations. Mutations alone probably do not play a causal role in aging, but they can lead to other biological changes and pathologies, including cancer development. On the other hand, the actual repair process leads to changes in chromatin structure, which can, in turn, make chromatin structure more prone to additional DNA damage. The main result of changes in chromatin structure, however, is deregulation of transcription, which may result in modification of tissue-specific expression patterns and thereby decrease the biological functions of tissues and lead to accelerated aging. Deregulated transcription can also impair DNA damage response machinery and chromatin structure maintenance, because specific expression profiles are needed for these processes to ensure maximum efficiency. Most importantly, it is not the effect of these individual processes alone, but rather their combination that might result in the aging phenotype.