The Role of Nuclear Architecture in Genomic Instability and Ageing

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The role of nuclear architecture in genomic instability and ageing

Philipp Oberdoerffer and David A. Sinclair Abstract | Eukaryotes come in many shapes and sizes, yet one thing that they all seem to share is a decline in vitality and health over time — a process known as ageing. If there are conserved causes of ageing, they may be traced back to common biological structures that are inherently difficult to maintain throughout life. One such structure is chromatin, the DNA–protein complex that stabilizes the genome and dictates gene expression. Studies in the budding yeast Saccharomyces cerevisiae have pointed to chromatin reorganization as a main contributor to ageing in that species, which raises the possibility that similar processes underlie ageing in more complex organisms.

Senescence Chromosomes are arguably the most difficult structures Although chromatin reorganization was linked to A nearly irreversible stage a cell has to maintain over a lifetime. The DNA in each ageing in budding yeast over 10 years ago8,9, these ideas of permanent G1 cell-cycle chromosome experiences thousands of chemical altera- have remained untested. Recently, a growing appre- arrest, which is linked to tions and DNA breaks in a single day, and the informa- ciation for the importance of chromatin in regulating morphological changes, metabolic changes and tion each encodes requires strict regulation to maintain gene expression and maintaining genomic integrity in changes in gene expression. cellular identity and function. To manage these tasks, complex organisms has reinvigorated interest in the link The induction of senescence eukaryotes have evolved a complex packaging system between chromatin alterations and ageing. In the past depends on p53 and cell-cycle known as chromatin, in which DNA is wrapped around 10 years, advances in nuclear imaging technologies have inhibitors such as p21 and p16. a protein core of four different histone dimers and forms a revealed a high level of chromatin organization that is nucleosome, the basic building block of chromatin. known as the nuclear architecture. In fact, genes from Recent studies have indicated that chromatin is a highly different chromosomes are often in close physical prox- dynamic form of nuclear organization that influences imity and form discrete foci dubbed transcription fac- DNA stability and gene-expression patterns1,2. The level tories, which help to orchestrate their transcription and of chromatin compaction can be modulated through organize the genome in the three-dimensional nuclear the chemical modification of histones (box 1) or of space (reviewed in Ref. 10). DNA. The more densely the nucleosomes are packed, The long-term maintenance of the nuclear archi- the more protected is the DNA from chromosomal tecture is vital for the normal functioning of cells damage3, but the less accessible it is for transcription2. and tissues over a lifetime. The dramatic effect of Highly compacted, transcriptionally silent chromatin a disturbed nuclear architecture is exemplified by is known as heterochromatin, whereas more accessible Hutchinson–Gilford progeria syndrome (HGPS), in chromatin is known as euchromatin (box 2). which a mutation that disrupts the nuclear architecture Unfortunately, the eukaryotic system of DNA leads to a disease with symptoms that resemble aspects Department of Pathology, packaging is not immune to the ravages of time. All of normal human ageing, such as loss of hair, restricted Paul F. Glenn Laboratories eukaryotes, including humans, experience changes in joint mobility and atherosclerosis11. Even cells from nor- for the Biological chromatin organization and gene-expression patterns mal individuals undergo significant nuclear architecture Mechanisms of Aging, 12 Harvard Medical School, as they age. In the late 1990s, a few researchers proposed changes in response to stress , and there are early hints 77 Avenue Louis Pasteur, that changes in chromatin organization underlie ageing- that normal human ageing is associated with alterations Boston, Massachusetts, USA. related changes in gene expression and the ageing in nuclear architecture13. Correspondence to D.S. process4,5. Changes in gene expression were already In this review, we discuss the causes and conse- e-mail: david_sinclair@hms. known to contribute to cellular senescence6, a possible quences of changes in nuclear architecture with age. harvard.edu 7 doi:10.1038/nrm2238 cause of ageing , and may provide an explanation for We focus on the role of epigenetic gene regulation dur- Published online the age-related decline in organ and tissue function in ing the ageing process, with an emphasis on drawing 15 August 2007 complex organisms. parallels between observations in yeast and mammals.

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Box 1 | Epigenetic silencing and the histone code

Epigenetic silencing describes heritable changes in gene Class I HDACs expression that do not involve modifications in coding Class III HDACs sequences. These changes include the methylation of Class II HDACs (sirtuins) HATs cytidine residues in genomic DNA or the modification of histones. Histones constitute the building blocks of a Remove Add protein core, around which DNA is wrapped to form a nucleosome, the fundamental packing unit of chromatin. These protein cores consist of two subunits of histones Me Me Ac Ac H2A, H2B, H3 and H4. Histone H1 is a linker histone that Me Me Ac Ac Ac helps to pack neighbouring nucleosomes together tightly75. Although most of each histone complex is inaccessible, histone tails protrude from the nucleosomes and are subject to post-translational modifications. These Add Remove modifications affect the secondary structure of histones and, thereby, the compaction of nucleosomes. Densely packed nucleosomes are generally inaccessible to the HMTs LSD1 transcription machinery and form epigenetically silent Jumonji family heterochromatin, whereas less compact nucleosomes are known as transcriptionally active euchromatin. Histone modifications include phosphorylation, methylation, acetylation, ubiquitylationNatur ande Re ADP-ribosylation.views | Molecular CeAllll of Biolog y these modifications are reversible, allowing for dynamic epigenetic gene regulation. Histone phosphorylation has mainly been associated with cell-cycle-mediated changes to chromatin and has a role in the DNA-damage response. Phosphorylated histone H2AX (or H2A in yeast) recruits the DNA-repair machinery and helps to resolve DNA double-strand breaks. The roles of ubiquitylation and ADP ribosylation are still not fully understood. Methylation and acetylation marks on histones are central to the regulation of the secondary structure of chromatin and for epigenetic silencing. Histone methylation is often associated with a repressed chromatin state, whereas acetylation generally coincides with transcriptionally active chromatin. Histone demethylases (such as the Jumonji family of enzymes) and histone acetyltransferases (HATs) are often involved in gene activation, whereas histone methyltransferases (HMTs) and histone deacetylases (HDACs) are important for the establishment and maintenance of heterochromatin, although there are exceptions (see figure). Changes in the expression levels or in the subnuclear localization of these enzymes (caused by DNA damage or over the lifetime of the organism) can be expected to affect nuclear architecture and gene-expression patterns (reviewed in Ref. 76). Ac, acetylation; LSD1, lysine-specific histone demethylase-1; Me, methylation.

We propose that a conserved DNA-damage response Sir2 mediates heterochromatin formation. One of the induces cumulative changes in chromatin structure and key regulators of yeast heterochromatin is Sir2 (silent nuclear architecture that are important driving forces information regulator-2), an NAD+-dependent histone behind the inexorable changes that occur in organisms deacetylase that predominately removes acetyl groups over time. These changes include a decline in genomic from Lys16 of histone H4 (Ref. 15). At the mating-type integrity, alterations in gene transcription and a loss of genes and telomeres, Sir2 interacts with its structural Mating-type locus (Refs 16,86) The mating of yeast only vitality — the series of changes we commonly refer to partners Sir3 and Sir4 , which regulate occurs between haploids, as ageing. and direct its deacetylase activity. Binding of the which can be either mating Sir4–Sir2 heterodimer to DNA nucleates DNA silencing type a or mating type α. The Heterochromatin alterations in yeast by recruiting Sir3 to form the Sir complex. Driven by mating type is determined by a single locus (MAT). Gene In the 1990s, a series of discoveries in the budding Sir2-dependent histone deacetylation, the Sir complex conversion between MAT and yeast Saccharomyces cerevisiae identified a mechanis- promotes heterochromatin formation by spreading the silent mating-type loci HML tic link between epigenetic silencing and ageing. The along chromatin through cycles of recruitment of other and HMR allows haploid yeast replicative age of a yeast cell is the number of offspring Sir complexes86. to switch to the active mating it produces before undergoing senescence (~23–30). At the rDNA locus, Sir2 is a crucial component of a type as often as every cell cycle. Like in all eukaryotes, heterochromatin in yeast serves network of protein–protein interactions that regulate two main purposes: it maintains certain genes (such as both silencing and DNA stability. In all eukaryotes, Telomeres the yeast mating-type loci) in a silent state through cell rDNA is organized as one or more arrays that contain Regions of highly repetitive division, and it stabilizes the highly repetitive parts 100–10,000 repeating units that are sequestered in the DNA at the ends of linear telomeres chromosomes. Telomeres of the genome (the and ribosomal DNA nucleolus. It is the highly repetitive nature of the rDNA function as caps to protect the (rDNA)), preventing them from recombining, fusing that renders it particularly susceptible to recombina- DNA ends from degradation or and breaking. Accordingly, yeast cells that lack crucial tion. The control of rDNA stability in yeast is well fusion with other heterochromatin factors are infertile because both understood. Yeast rDNA is silenced and stabilized by chromosomes, and as types of mating-type genes are expressed concurrently. the regulator of nucleolar silencing and telophase exit facilitators of DNA replication at the ends of chromosomes Furthermore, telomeres become extremely short and (RENT) complex, which consists of Sir2, Net1 and 17 by recruiting the reverse tend to fuse, which causes major problems during cell Cdc14 in a 1:1:1 ratio. Net1 recruits Sir2 to the rDNA , transcriptase telomerase. division (reviewed in Ref. 14). where it forms a complex with, and thereby sequesters,

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Box 2 | Constitutive versus facultative heterochromatin that is associated with the relocalization of Sir3 to the nucleolus, which becomes dramatically enlarged and There are two types of heterochromatin: constitutive and facultative (inducible). fragmented23 (FIG. 1a). Taken together, these findings Although both types can be stably inherited over numerous cell divisions, each type suggest that the relocalization of chromatin-modifying has a distinct make-up and function. Constitutive heterochromatin mainly comprises proteins is a normal event during yeast ageing repetitive genetic elements, such as telomeres and centromeres, that localize to the nuclear periphery. By contrast, facultative heterochromatin can form anywhere in the and can have a dramatic effect on genomic stability and nucleus and its formation is required for mating-type gene silencing in budding yeast, lifespan. and X‑chromosome inactivation and developmental progression in mammalian cells The yeast findings raised an intriguing question: (reviewed in Ref. 77). how does the nucleolus affect ageing? In 1997, ageing Both constitutive and facultative heterochromatin form repressive chromatin of yeast was shown to stem from the inherent instabil- structures and are associated with transcriptional silencing. Silencing by constitutive ity of rDNA9. rDNA is highly repetitive and therefore heterochromatin is considered to be indirect and rather unspecific in nature. prone to homologous recombination. Recombination Constitutive heterochromatin is thought to act primarily as a genome stabilizer that between rDNA sequences results in the excision of a prevents gene rearrangements between highly similar genetic sequences and ensures single circular molecule of DNA (an extrachromosomal efficient chromosomal segregation. As a by‑product, genes that are adjacent to rDNA circle (ERC)) that is replicated during S phase. repetitive DNA can be silenced by the spreading of silent heterochromatin into non-repetitive, gene-containing regions — this phenomenon is known as Roughly 12 divisions later, the nucleus becomes packed position-effect variegation78. with >1,000 ERCs that cause cell death, presumably by 9 Facultative heterochromatin is often localized to promoters and is established either titrating essential proteins from the rest of the genome . in a developmentally regulated manner or in response to environmental triggers. In young yeast cells, these recombination events are It ensures the epigenetic silencing of genes in a certain cell type or tissue, and is an controlled by Sir2, which directly binds to the rDNA important mechanism for developmental programming and cell fate. Initiation of this and deacetylates the surrounding histones, resulting in form of heterochromatin depends on site-specific transcriptional repressors, which, chromatin compaction and rDNA stabilization. in turn, recruit a silencing complex that contains histone-modifying enzymes and other This discovery provided a direct link between hetero- structural proteins that maintain a silent state. Stress-induced facultative chromatin and ageing and led to a testable prediction. heterochromatin can be the consequence of cellular stress such as the induction of Decreasing heterochromatin at the rDNA locus should senescence. Its formation requires nuclear factors that are involved in cell-cycle progression and, possibly, the recruitment of the DNA-repair machinery12,42. accelerate ageing, whereas increasing it should extend lifespan. This hypothesis was confirmed by manipulating SIR2: deletion of the SIR2 gene led to a loss of rDNA silencing, elevated rDNA recombination and accelerated the Cdc14 phosphatase. Cdc14 is involved in cell-cycle ageing, whereas integration of an extra copy of the regulation and is kept inactive while in the RENT com- SIR2 gene increased rDNA silencing, suppressed rDNA plex18,19. Whether it also has a role in rDNA silencing recombination and extended lifespan by 30%17. remains unclear. The first clear genetic link between heterochromatic Heterochromatin reorganizes in response to DNA damage. silencing and ageing came from a genetic screen that Why does Sir protein redistribution occur during ageing? Progeroid disease A genetic disorder in which isolated a gain-of-function mutation in SIR4, known as The answer may come from the growing appreciation of various tissues, organs or SIR4-42 (Refs 8,20). SIR4-42 generates a truncated Sir4 the importance of chromatin in maintaining genomic systems of the human body protein, which cannot bind to telomeres and mating- stability and facilitating DNA repair. During the DNA- appear to age prematurely. type genes, thus abolishing silencing at these loci. The repair process, chromatin needs to be unpacked and These diseases are often called mutant Sir4 protein targets a greater amount of Sir2 and reassembled, which has an important influence on the segmental progeroid diseases because they do not fully Sir3 to the nucleolus, which, in turn, correlates with an rate and type of repair. One possibility is that the relo- recapitulate normal ageing. A increase in mean lifespan by ~40%. Thus, recruitment calization of Sir proteins is an active defence process common feature of such of Sir4 to the nucleolus early in life seems to slow age- that the cell initiates to stabilize its DNA. diseases is genomic instability. ing and extend lifespan. Examination of wild-type yeast In 1999, four studies showed that a single DNA

RecQ DNA helicase cells showed that the redistribution of Sir proteins to break is sufficient to illicit a DNA-damage-checkpoint One of a family of DNA the nucleolus is not limited to mutant Sir4, but reflects response that releases Sir proteins from mating-type helicases that help to stabilize a normal process during yeast ageing8, albeit one that loci and telomeres and relocalizes them to the DNA replication forks and remove occurs later in life than in a SIR4-42 mutant. break, possibly to facilitate the repair process24–27. DNA recombination Around the same time as the characterization of Indeed, genomic DNA from SIR2 mutants was shown intermediates, thereby maintaining genome integrity. SIR4-42, a study of the yeast WRN homologue also to be more susceptible to cutting by an endogenously 25 In humans, there are five family pointed to the nucleolus as an important site that influ- expressed EcoRI endonuclease . However, no defect in members; mutations in three of ences ageing. In humans, loss-of-function mutations in DNA end-joining was observed using a plasmid-based these helicases are associated the human WRN gene cause Werner syndrome (WS), a assay26, a discrepancy that may be explained by the lack with a predisposition to cancer progeroid disease and premature ageing. that mimics many aspects of normal of chromatin on plasmid DNA. This finding fits with ageing including atherosclerosis, diabetes and dramati- the observation of Tyler and colleagues, who found Position-effect variegation cally aged skin by age 40. WRN and its yeast homologue that Sir2 and other histone deacetylases modify the The variation in gene SGS1 encode RecQ DNA helicases that function in DNA chromatin that surrounds the break site in a temporally expression that can occur repair and recombination21,22. In the absence of RecQ coordinated manner, which appears to be a prerequisite between genetically identical 28 cells when a gene is juxtaposed helicases, genomes are highly unstable, especially of efficient repair . We refer to this process as the to a region of contracting and at repetitive loci. Deletion of SGS1 results in hyper- relocalization of chromatin-modifying factors (RCM) expanding heterochromatin. recombination at the rDNA and premature ageing response (FIG. 1a).

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The damage-mediated relocalization of Sir proteins RCM response, alterations in silencing and irreversible appears to have little effect on long-term genomic genomic changes. A role for DNA damage in age-related silencing patterns in young yeast cells. However, the genomic instability has also been reported for loci other accumulation of DNA damage and increased rDNA than the rDNA. DNA-break-induced loss of heterozygo instability in old yeast cells eventually leads to a chronic sity at artificially generated heterozygous loci was found

a Yeast Accelerated ageing SGS1 mutant Genotoxic stress, DNA damage replication MATa Chromosomes MATα ERC

rDNA Normal ageing

Genotoxic stress, Accumulation replication of ERCs

b Human Perinuclear Hutchinson–Gilford heterochromatin progeria Transcriptional Defective deregulation lamin A

Facultative heterochromatin Euchromatin Normal ageing Environmental Changes in stress, nuclear DNA damage architecture

SAHFs

Age Figure 1 | Comparison of age-related changes in nuclear architecture between yeast and mammalian cells. a | Schematic of accelerated ageing (top) and normal ageing (bottom) in a replicatingNa yeastture nucleus. Reviews |In Mol youngecular yeast Cell cells,Biolog y telomeres, mating-type loci (MATa and MATα) and ribosomal DNA (rDNA; orange) are silenced by silent information regulator-2 (Sir2)-containing complexes (purple circles). In wild-type yeast, homologous recombination at the highly repetitive rDNA locus generates extrachromosomal rDNA circles (ERCs) during cell division. Lack of the DNA helicase Sgs1 causes genomic instability at the rDNA and leads to increased ERC formation, accelerated changes in nuclear architecture and premature ageing23 (top). Sites of DNA damage and both ERCs and rDNA recruit components of the Sir2- silencing complex, causing a loss of silencing at telomeres and mating-type loci (green represents areas of transcriptional derepression). b | Changes in nuclear architecture of human cells in a model of accelerated ageing (top) and normal ageing (bottom). Young cells show dense, transcriptionally inaccessible perinuclear heterochromatin surrounding less densely packed, transcriptionally active euchromatin. Grey circles represent sites of facultative heterochromatin and blue ovals depict constitutive, perinuclear heterochromatin (BOX 2). In Hutchinson–Gilford progeria syndrome (HGPS), a defect in the nuclear lamina component lamin A leads to an accelerated loss of pericentromeric heterochromatin and concomitant changes in nuclear architecture that are accompanied by transcriptional deregulation13,58,59 (green areas). Similar changes have been observed during normal ageing. Cellular stress can cause the formation of repressed senescence-associated heterochromatin foci (SAHFs)12.

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to increase dramatically with age and, depending on the The contribution of this effect on AT pathology remains locus, was aggravated in the absence of Sir2 (Ref. 29), unclear and is complicated by the range of defects that again indicating a protective role for heterochromatin. are observed in ATM-defective cells. The yeast ATM Both increased susceptibility to DNA damage and an orthologue Tel1 has also been linked to telomere main- accumulation of defective DNA-repair enzymes might tenance36, which further suggests that pathways that are explain how ageing can promote this global genomic involved in the maintenance of nuclear architecture instability. are highly conserved.

Nuclear changes in ageing mammals Chromatin-structure changes and epigenetic silencing. How do these findings in yeast relate to what is known Changes in nuclear architecture do not appear to be about the role of heterochromatin and epigenetic silenc- restricted to defects in the structural components of the ing in mammals? Although the human genome and nucleus. An age-related loss of epigenetic silencing at human ageing exceed the yeast model in complexity, the certain repetitive elements was reported almost 20 years yeast studies may help us to understand fundamental ago. Specifically, the major satellite repeats that form processes that govern conserved aspects of ageing. heterochromatic chromatin structures around the cen- Indeed, changes in heterochromatin composition and tromeres of every chromosome were shown to be more structure with age have been reported in several species transcriptionally active in aged cardiac tissue, which sug- including humans. gests a progressive loss of silencing of these elements37. Given the number of repetitive elements in mammalian Lessons from human progeroid syndromes. WS, HGPS genomes, a reduction in repeat-associated heterochro- and ataxia telangiectasia (AT) are rare genetic premature matin would be consistent with significant changes in ageing disorders that demonstrate both the dramatic nuclear architecture. Shen et al. recently reported a pos- consequences of defects in nuclear architecture and the sible mechanistic link between mammalian ageing and diverse sets of genes that are involved in its maintenance. changes in heterochromatin38. Older individuals show As mentioned above, WS is characterized by a genome altered activity in their histone-modifying enzymes, that is highly unstable owing to the lack of functional which causes a loss of perinuclear heterochromatin RecQ helicase22. HGPS shows similarities to WS but and concomitant changes in gene expression. These proceeds more rapidly11. One known cause of HGPS is observations are reminiscent of the chromatin changes a single base change in the LMNA gene, which encodes that occur during yeast ageing and in HGPS, and raise lamin A, an essential structural component of the the possibility that changes in perinuclear architecture nuclear membrane30. The mutant LMNA gene generates contribute to normal ageing in mammals (FIG. 1b). a truncated splice variant that disturbs the structure of Numerous other epigenetic changes in nuclear archi- the nuclear membrane and causes large changes in the tecture and gene expression have been associated with nuclear architecture. Nuclei from patients with HGPS ageing. More than a decade ago, Imai and colleagues showed are characterized by a dysmorphic shape and a loss of that collagenase, a gene associated with cellular ageing, is heterochromatin-related proteins that are associated differentially regulated during cellular senescence — a with the nuclear membrane, such as heterochromatin phenomenon that is often referred to as cellular ageing39. protein-1 (HP1), as well as altered histone-modification This effect appears to be due to changes in the subnuclear patterns that reflect a general disturbance in silent localization of the collagenase gene as cells undergo senes- heterochromatin (FIG. 1b). Interference with aberrant cence. In young cells, the collagenase gene is repressed by LMNA splicing can reverse the structural defects that the transcription factor OCT1. A considerable proportion are typical for HGPS in cell culture, which demonstrates of OCT1 was found in the heterochromatic nuclear a direct causal relationship between the LMNA gene and periphery, where it colocalized with lamin B, a component HGPS31. The truncated LMNA splice variant has also of the nuclear membrane. This interaction was abrogated been found in naturally old humans, which implicates in senescent cells and, concomitantly, collagenase repres- changes in lamin A in the normal ageing process13. A sion was lost39. On the basis of these findings, the authors conserved role for lamin A in the ageing process is con- proposed a model of age-associated heterochromatin sistent with the recent finding that neuronal cells in the reorganization that would account for such transcriptional nematode Caenorhabditis elegans also show changes in changes in a global manner5. the nuclear architecture in aged animals, and a loss-of- This idea gained support from recent studies of cell function mutation in the worm orthologue of lamin A ular senescence, most notably by Lowe and colleagues, causes a decrease in life expectancy32. who found that senescence is associated with an overall Like WS and HGPS, AT is characterized by a defective increase in non-pericentromeric, facultative heterochro- nuclear architecture, progressive neurological degenera- matin domains, known as senescence-associated hetero- tion, growth retardation, genomic instability and prema- chromatin foci (SAHFs; FIG. 1b)12. SAHFs form repressive ture ageing33. Patients with AT have an inherited defect chromatin structures that can be found at, but are not lim- in the AT mutated (ATM) gene, which encodes a protein ited to, promoter regions of certain cell-cycle regulators, kinase that initiates the DNA-repair cascade. DNA repair in particular target promoters of the cell-cycle regula- and ATM in particular seem to be required for telomere tor E2F. This finding led to the hypothesis that SAHFs maintenance34,35, and defective ATM can disturb the promote senescence through direct repression of growth- interactions between telomeres and the nuclear matrix34. promoting genes. Although the repression of cell-cycle

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Table 1 | A comparison of age-related changes in gene expression between species Significantly altered Homo sapiens Macaca Mus musculus Drosophila Caenorhabditis functional groups mulatta melanogaster elegans Cortex43 Muscle73 Muscle46 Cortex44 Muscle45 Whole body49 Whole body85 Stress response ↑ ↑/↓ ↑/↓ ↑ ↑ ↑ ↓ Hormonal genes and ↑/↓ ↓ ↓ ↑ growth regulators Inflammation and ↑ ↑ ↑ ↑ immune response Metabolism ↑ ↓ ↓ ↑ ↓ ↑/↓ ↑/↓ Transcription ↑/↓ ↑/↓ ↓ Biosynthesis ↓ ↓ Protein turnover ↑/↓ ↑ ↓ ↓ Signalling ↓ ↑ Developmental ↓ ↑/↓ process Vesicular transport ↓ ↑ Survival ↓ Mitochondrial genes ↓ ↓ ↓ Neuronal factors ↓ ↑ ↑ RNA binding and ↑ processing Other deregulated 67% 87% 78% 50% 60% 80% NS genes* Upregulated genes NS 55% 67% 59% 51% 62% ~60% NS, not specified. *Genes that did not fall into any of the categories highlighted by the authors.

regulators is an important function of SAHFs during a phenomenon reminiscent of the RCM response that cellular senescence, the frequency and distribution of occurs in yeast in response to DNA damage and ageing. these foci suggests a much broader impact of SAHFs. However, it is important to keep in mind that cellular This notion is further supported by the finding that the senescence — although it is a likely contributor to cancer formation of SAHFs appears to rely on the recruitment of and organismal ageing — does not equal the complex proteins from promyelocytic leukaemia nuclear bodies40, physiological processes that, together, define what is which have been implicated in numerous cellular pro- called ageing. The relevance of the aforementioned cesses including transcriptional regulation, apoptosis findings to the functional decline of higher organisms and cellular defence in response to stress (most notably remains to be elucidated. to DNA damage41). A connection between senescence-associated het- Changes in epigenetic gene regulation erochromatin formation and mammalian ageing has In yeast, the redistribution of chromatin-modifying recently been made using baboon skin fibroblasts42. enzymes to the rDNA destabilizes telomeres and exposes Tissue from older individuals accumulates cells contain- them to degradation, and desilences the mating-type loci, ing heterochromatic foci that are reminiscent of SAHFs causing sterility. In mammals, ageing has been associated in senescent cells. These foci were found in >15% of the with large-scale changes in both nuclear architecture and total cell population in aged tissues, which suggests that chromatin structure. How might these changes contribute a significant fraction of aged tissue may be expressing to the ageing process? Because numerous genes are either markers of senescence and undergoing large-scale directly or indirectly regulated by (nearby) heterochro- heterochromatic changes. Importantly, the emergence matic regions2, it is possible that changes in the epigenetic of heterochromatin foci occurred simultaneously with make-up of a cell might alter its gene-expression patterns, telomere shortening, which points to a shift from stable, thereby changing its genomic identity. In this section, we perinuclear heterochromatin to induced, or facultative, discuss evidence for age-related changes in gene expres- heterochromatin. These studies raise the intriguing possi sion across species and a possible role for DNA damage as bility that the age-associated loss of genomic silencing an evolutionarily conserved mechanism that could drive detected in previous studies may be linked to, or caused changes in nuclear architecture and gene expression over by, the formation of SAHF-like heterochromatic foci, a lifetime.

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Gene expression changes with age. With the emergence merely a consequence thereof. Findings in yeast suggest of genomic technologies, age-associated alterations in that there may be a causal link: Sir2 not only facilitates gene-expression patterns have now been documented in heterochromatin and promotes DNA stability, but is several species (TABLE 1). An impressive example of age- also a mediator of calorie restriction51,52. Furthermore, related alterations in gene expression comes from a study in rodents and humans, the levels and activity of the by Yankner and colleagues43. The authors examined Sir2 orthologue SIRT1 increase in response to calorie gene-expression patterns in the human cortex, covering restriction53, which raises the possibility that the enzyme a broad age range, and observed progressive changes in may also be involved in age-related changes in nuclear gene-expression patterns with age. Comparisons between architecture and could be a mediator of caloric restric- age-related gene-expression patterns across tissues and tion in mammals. It will be interesting to explore to what even species reveals that several functional gene groups extent SIRT1 directly regulates gene expression (so far, are similarly affected; the increased expression of stress- only a few examples are known54–56) and whether SIRT1 response genes and inflammatory genes is one example facilitates heterochromatin formation or promotes (TABLE 1). Such changes may reflect a response to age- genomic stability in mammals. If so, perhaps some of related stress and are thought to counteract age-related the age-related changes in gene expression and genomic tissue damage. instability in mammals can be traced to the relocaliza- Could some of the reported transcriptional changes tion of SIRT1 during ageing, as is the case for the yeast be a cause rather than a common consequence of age- orthologue Sir2. ing? The majority of significantly altered transcripts cover a broad and seemingly random range of genes, DNA damage causes genome-wide transcriptional some of which may interfere with proper cell function; changes. Why gene-expression patterns change during examples include the deregulation of cell-cycle genes in ageing is not known; however, it has been speculated that post-mitotic neurons44 and neuronal factors in muscle a major underlying cause of these changes is DNA dam- tissues45,46. Such changes may pose a problem for proper age (box 3). Yankner and colleagues first demonstrated cell function and thereby directly contribute to organ a direct link between global age-related gene repression decline and ageing. In addition, although there are and oxidative DNA damage to the promoters of the groups of genes that are shared between species, most repressed genes43. Oxidative DNA damage is caused by age-related transcriptional changes are not shared, even an accumulation of reactive oxygen species (ROS), which between closely-related species such as monkeys and can be observed with age. ROS are highly unstable, humans47. Even within species, the majority of trans reactive by-products of mitochondrial respiration and criptional changes appear to differ between tissues48. can damage several cellular components including lipids, If age-related transcriptional changes were solely a proteins and DNA. Oxidative stress has, therefore, been consequence of the ageing process, one might expect proposed to be a significant contributor to cellular and similar changes between species and, certainly, organs organismal decline and its role during ageing has been of the same animal. The observed variation between extensively investigated (reviewed in Ref. 57). The idea species and tissues underlines the apparent randomness that increased ROS generation may be responsible for of these changes. A stochastic component that affects gene-expression changes is further supported by com- gene-expression changes is also supported at the level parisons of cerebellar and cortical gene-expression of individual cells, as transcriptional profiles can differ patterns of aged monkeys and humans. The tissue with between adjacent cells from the same aged tissue of higher respiratory activity and presumably higher pro- a mouse49. However, the combined transcriptional pensity for DNA damage, in this case the cortex, showed changes within a given tissue appear to be rather repro- greater alterations in gene regulation with age47. However, ducible, which implies that the pre-existing nuclear more work is needed before a causal relationship environment of a cell or tissue may also determine can be declared between respiration, DNA damage and which genes become preferentially deregulated with age. gene-expression changes in the brain. This hypothesis is further supported by work in flies, As previously mentioned, gene expression is not only which demonstrates that some genes that are deregu- altered during ageing in mice, but can vary between lated during ageing localize to the same chromosomal single cells in a homogeneous tissue. These changes region; this finding indicates a role for global changes in can be accelerated by oxidative DNA damage in cell- nuclear architecture with ageing50. Clearly, more work culture experiments49. This effect on gene expression is required to understand the underlying causes of age- was long-lasting, persisting up to 9 days after stress related transcriptional changes and their contributory — a finding that is reminiscent of long-lasting stress- relationship, if any, to the ageing process. induced changes in chromatin structure12. This study in particular supports the idea that randomly distributed Calorie restriction counters gene-expression changes. sites of DNA damage can influence gene expression Calorie restriction, a dietary regimen that extends with age. It also implies that much of the variability in the lifespan of numerous organisms, also delays the transcription among single cells cannot be detected by majority of age-related gene-expression changes in whole-tissue analyses. Given that most of the studies mice and, to a certain extent, in flies45,50. It is currently so far have examined whole tissues, the number of unclear whether the effect of calorie restriction on gene genes that are deregulated with age is likely to have been expression underlies its beneficial effect on lifespan or is underestimated.

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60 Box 3 | DNA damage and ageing gene expression changes : Hoeijmaker’s group identified a novel mutation in a member of the well characterized The DNA in the genomes of all organisms can be altered by exposure to numerous xeroderma pigmentosa (XP) complementation group, XPF. environmental agents, such as ultraviolet (UV) or other radiation and chemical XPF is part of an endonuclease complex that is involved carcinogens, and also by cellular processes that lead to DNA alterations — for example, in the repair of single-nucleotide lesions and DNA inter- erroneous DNA-replication and free radicals such as reactive oxygen species (ROS), strand crosslinks. Humans with this particular mutation which emerge as toxic by-products of mitochondrial respiration (see figure). Because DNA-repair mechanisms are imperfect, the amount of damaged DNA inevitably increases show dramatic progeroid symptoms and usually die in with the age of the organism. Depending on the sites of damage, the consequences can their teens. A mouse model for this disease shows gene- range from innocuous to deleterious. For example, mutations in tumour-suppressor expression patterns that resemble those of normally aged genes can promote tumorigenesis, and expansion of the glutamine-coding CAG mice, and metabolic gene-expression changes appear trinucleotide during repair of oxidatively damaged DNA can result in to be particularly conserved. The authors suggest that neurodegeneration79. this may reflect a common stress response that provides Although these age-related diseases are strongly influenced by DNA damage, there is protective tissue maintenance. Although this correlation still much debate about the extent to which DNA damage contributes to ageing. On the was impressive, a significant number of other ageing-like one hand, there is a clear link between oxidative stress and lifespan in invertebrates. transcriptional changes that were reported for this XPF In mammals, calorie restriction — a dietary intervention known to extend lifespan — mouse model do not fall into stress-response categories. reduces ROS production and increases the expression of enzymes that metabolize ROS, Together, these observations suggest that, although some such as superoxide dismutases (SODs) and catalase (reviewed in Ref. 80) (see figure). Decreased DNA damage and increased lifespan have also been observed in mice that of the age-related transcriptional changes constitute a overexpress catalase in mitochondria81. Similarly, mice with mutations in DNA-repair stress response, a significant proportion of the changes enzymes that are involved in transcription-coupled repair or base-excision repair show occurs in a seemingly random fashion. signs of premature ageing60,82. In humans, several defective DNA-repair pathways can cause accelerated ageing (progeroid) syndromes. On the other hand, certain mouse DNA damage alters chromatin strains with defective DNA-repair systems accumulate high levels of DNA damage and In yeast, DNA damage induces an RCM response that yet have a normal lifespan (reviewed in Ref. 83). Similarly, a reduction in SOD levels in disrupts heterochromatin and alters gene expression. 84 mice leads to increased oxidative DNA damage but does not affect the ageing process . A wealth of literature has recently implicated chroma- Recent work suggests that certain types of DNA damage can significantly alter the tin-remodelling enzymes in the DNA-repair process gene-expression profile of an organ and that these changes — rather than DNA damage directly — might be the cause of organ decline and ageing60. Little is known about how in yeast and other more complex organisms (reviewed in defective DNA repair or increased oxidative stress may cause such global gene- Ref. 61). DNA double-strand break (DSB) repair involves expression changes, and more work is needed to fully understand the role of DNA the recruitment of histone modifiers to the repair site, damage in ageing. MnSOD, manganese superoxide dismutase. together with other repair complex components such as Ku70/80 and DNA ligase IV. DSBs trigger the DNA- Chemical carcinogens, damage sensor kinases ATM, ATR or DNA-PK, which UV and other Lipids phosphorylate the surrounding histones H2A and H2AX radiation Caloric restriction in yeast and mammals, respectively62–64. The extent of this modification can reach into megabases, potentially 65 Proteins ROS affecting the epigenetic regulation of several genes . MnSOD In yeast, chromatin-remodelling factors that are Mitochondrial respiration involved in DNA repair, such as histone acetyltrans- SOD1,2; ROS ferases (HATs) and histone deacetylases (HDACs) as catalase well as histone methyltransferases, are then recruited to Cell membrane 28,66,67 Mitochondria the break site . Although the precise role for these ROS chromatin-remodelling complexes during DNA repair is not fully understood, it is presumed that they dictate the type of repair and facilitate the repair process by changing DNA-repair DNA-repair the chromatin composition around the site of damage. factors factors In yeast, HATs (such as Esa1) and HDACs (in particular CAG Sin3, Rpd3 and the sirtuin Hst1) are part of chromatin- Changes in gene Repeat expansion Mutations in remodelling complexes that promote an ordered and expression tumour suppressor dynamic progression of histone modifications over the time-course of DSB repair28. Importantly, these proteins Nature Reviews | Molecular Cell Biology are not specialized DNA-repair enzymes but, rather, The fact that DNA repair is impaired in mouse chromatin-modifying enzymes that have several func- models of HPGS suggests that DNA damage also has a tions outside DNA repair, including gene silencing68. Transcription-coupled repair 58 A DNA-repair mechanism that role in this premature ageing syndrome . cDNA micro- Recruitment of such factors to sites of damage may operates in tandem with array analysis of fibroblasts from patients with HGPS therefore be accompanied by a loss of function at their transcription and involves showed a range of gene-expression changes that cover original sites, as is the case for the Sir complex25,27. members of the XP gene family. gene-ontology groups as diverse as signal transduction, Although a role for histone deacetylation during Failure of the transcription- transcriptional regulation, cell-cycle regulation and DNA repair has not been demonstrated in mammalian coupled repair mechanism results in Cockayne syndrome, development, consistent with a global deregulation of cells, there is accumulating evidence for chromatin 59 an extreme form of accelerated gene expression . A recent report further highlighted remodelling besides H2AX phosphorylation. For example, ageing that is fatal early in life. the dramatic effect of DNA-repair defects on age-related histone methylation has been directly linked to the

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Young appears to be evolutionarily conserved between yeast and Perinuclear mammals because the fission yeast 53BP1 homologue heterochromatin Crb2 is also recruited to DSBs, a process that requires methylation of Lys20 on H4 (Ref. 66). The fact that 53BP1 Facultative can also be found in the DNA-damage-associated hetero heterochromatin chromatin foci of aged monkey fibroblasts42 further Euchromatin corroborates the idea that histone modifiers may have a crucial role during the DNA-damage response in mammals and points towards DNA damage as an inducer of global changes in chromatin architecture.

The epigenetic balance hypothesis As mentioned earlier, the formation of transient heterochromatic foci around sites of DNA damage may Gene A Gene B Gene C explain how DNA damage might directly mediate gene repression12. Consistent with this notion, many of the gene-expression changes that are observed in aged indi- Old viduals occur in a stochastic fashion, as does most DNA Loss of perinuclear damage49,71. It is also conceivable that certain genomic heterochromatin regions are more prone to damage than others, which DNA damage could explain some of the predictable, co‑regulated changes that are observed between aged individuals Facultative heterochromatin of the same species. Indeed, DNA breaks occur more stress-induced commonly in certain euchromatic, active regions of the SAHFs genome3. Furthermore, fragile sites on chromosomes that are prone to breakage are well documented in mam- mals72. It will be interesting to investigate whether these sites of preferential DNA damage correlate with loci that become deregulated with age. Although it is conceivable how DNA damage might lead to gene repression, it is less obvious how age-related Gene A Gene B Gene C stress and DNA damage could account for gene activation. This question is particularly important to address Figure 2 | Redistribution of heterochromatin-associated factors as a cause of because the fraction of genes that are significantly age-related changes in nuclear architecture andNa geneture Re expression.views | Molecular In the Ce young,ll Biolog y upregulated with age roughly equals or even exceeds the nuclear architecture of each tissue is well defined; it comprises tightly packed the fraction that is downregulated (TABLE 1). Moreover, perinuclear heterochromatin (blue) and patches of tissue-specific, developmentally transcript levels overall appear to be increased in old controlled facultative heterochromatin islands (grey; represented by gene A) in the animals73. One explanation may be that DNA damage otherwise transcriptionally active euchromatin (represented by gene B). This generates cell-type-specific gene-expression patterns. Repetitive DNA is part of perinuclear interferes with the expression of transcriptional repres- heterochromatin and is transcriptionally repressed. Position-effect variegation (BOX 2) sors, leading indirectly to an induction of sets of target can cause repression of nearby coding regions (gene C). Silencing complexes in genes. However, the diversity of genes that show increased constitutive and facultative heterochromatin are different (grey and blue ovals), but expression with age indicates that other processes contain several identical chromatin-modifying enzymes (green ovals). Green tags may also be at work. represent transcriptionally permissive histone modifications, whereas red tags represent Based on what we know about yeast ageing and the non-permissive histone modifications. The age-associated accumulation of DNA damage DNA-damage-induced RCM response, we propose triggers global changes in nuclear architecture, including the formation of senescence- the following model for how DNA damage might lead associated heterochromatin foci (SAHFs) in euchromatic DNA (grey) and a gradual loss of to global changes in gene expression to promote ageing perinuclear heterochromatin. This loss may be a direct consequence of a redistribution of in mammals. We refer to this model as the ‘epigenetic essential silencing factors, in particular histone-modifying enzymes, to sites of DNA (FIG. 2) damage or SAHFs (arrows). This process causes changes in nuclear architecture and balance hypothesis’ . In this model, age-related tissue-specific gene expression patterns. In this example, gene A is activated owing to gene-expression changes are manifestations of the the loss of facultative heterochromatin, gene B is silenced in response to DNA damage redistribution of chromatin modifiers from one genomic and gene C is derepressed owing to changes in position-effect variegation. The locus to another. The model also encompasses the idea corresponding changes in histone modifications are shown. that DNA damage mediates chromatin remodelling and changes in nuclear architecture that occur over a lifetime, which fits with evidence that oxidative stress and DNA damage can accelerate the ageing process. Based on the recruitment of DNA-repair factors in human cells. observations of Tyler and colleagues28, it is plausible that Specifically, methylation of Lys79 on histone H3 recruits chromatin modifications during DNA repair are never the p53-binding protein-1 (53BP1). Recruitment of these fully restored to their pre-damaged state, resulting in factors is thought to tether the DNA-repair complex to progressive alterations in both chromatin-modification the site of damage69,70. Importantly, this mechanism patterns and gene expression28.

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Base-excision repair The consequences of the redistribution of chromatin- which in turn enhances susceptibility to DNA damage. (BER). A DNA-repair pathway modifying enzymes would be twofold. Previously silent In this scenario, gene-expression changes would precede that corrects single mutated regions may become transcriptionally active, leading to DNA damage. Despite convincing evidence for DNA bases. The two main enzymes ectopic gene expression and, possibly, destabilization of damage as a trigger of transcriptional changes49,60, it is used in BER are DNA glycosylases and apurinic or previously heterochromatic repetitive DNA. By contrast, conceivable that a change in the transcription status of apyrimidinic (AP) genes may become repressed near sites of DNA damage a gene determines its susceptibility to DNA damage. A endonucleases. The DNA through remodelling processes that are similar to the comprehensive (computational) analysis or the genome- glycosylase hydrolyses the formation of SAHFs. According to the model, nuclear wide mapping of sites of DNA damage and localization glycosidic bond to create an structure and organization is progressively and inexor of chromatin-remodelling enzymes may shed light on the AP site, which is then recognized and excised by the ably altered over time, resulting in the functional decline complex interplay between transcriptional activity and AP endonuclease, allowing of cells and tissues. The model is consistent with both DNA damage. DNA polymerases to replace the stochastic changes in gene expression as described Several findings suggest that DNA damage is a main the missing base. by Vijg and colleagues49 and the reproducible tissue- trigger of nuclear ageing, supporting the free-radical 74 Xeroderma pigmentosa specific transcriptional changes that occur as organisms theory of ageing (see the accompanying Opinion article A genetic DNA-repair disorder age, which will be dictated by the original architecture by Pelicci and colleagues in this issue). However, it could in which the ability of the body of a given tissue. also be argued that chromatin structure is directly affected to remove damage caused by by the ageing process through an as-yet-unknown mecha- ultraviolet light is impaired, Perspective nism that leads to increased DNA damage and a perma- leading to multiple basaliomas and other skin malignancies at In this review, we propose that a redistribution of chro- nent damage response that alters gene-expression patterns a young age. matin modifiers is a natural, protective response to in a similar way to the model proposed in this review. DNA damage, but may lead to epigenetic changes that Over the coming years, as researchers use mammalian affect genomic integrity and, thereby (at least in part), models to map the global pattern of chromatin modifi- account for changes in gene expression that appear to be cations during ageing, it should become clear whether a hallmark of the ageing process. Although this epigenetic changes in the epigenetic balance due to the RCM balance hypothesis presents an appealing explanation of response underlie aspects of the ageing process. For now, what we currently know about age-related changes in perhaps we should pause for a moment to consider the nuclear architecture and gene expression, it is certainly remarkable ability of cells to maintain their chromatin not the only way to explain the observed effects of age- and gene-expression patterns for as long as they do, ing. For example, it can be argued that changes in gene overcoming daily chemical and physical damage, in some expression mediate changes in chromatin structure, cases for many decades.

1. Cheutin, T. et al. Maintenance of stable 13. Scaffidi, P. & Misteli, T. Lamin A‑dependent nuclear telomeric silencing and Mec1p-dependent relocation heterochromatin domains by dynamic HP1 binding. defects in human aging. Science 312, 1059–1063 of Sir3p. Curr. Biol. 9, 963–966 (1999). Science 299, 721–725 (2003). (2006). 25. Mills, K. D., Sinclair, D. A. & Guarente, L. MEC1- 2. Grewal, S. I. & Jia, S. Heterochromatin revisited. Implicates the main cause of HGPS — a defective dependent redistribution of the Sir3 silencing protein Nature Rev. Genet. 8, 35–46 (2007). lamin A splice variant — in normal human ageing. from telomeres to DNA double-strand breaks. Cell 97, 3. Obe, G. et al. Chromosomal aberrations: formation, 14. Sinclair, D. A., Mills, K. & Guarente, L. Molecular 609–620 (1999). identification and distribution. Mutat. Res. 504, mechanisms of yeast aging. Trends Biochem. Sci. 23, 26. Lee, S. E., Paques, F., Sylvan, J. & Haber, J. E. Role of 17–36 (2002). 131–134 (1998). yeast SIR genes and mating type in directing DNA 4. Villeponteau, B. The heterochromatin loss model of 15. Imai, S., Armstrong, C. M., Kaeberlein, M. & double-strand breaks to homologous and non- aging. Exp. Gerontol. 32, 383–394 (1997). Guarente, L. Transcriptional silencing and longevity homologous repair paths. Curr. Biol. 9, 767–770 5. Imai, S. & Kitano, H. Heterochromatin islands and protein Sir2 is an NAD-dependent histone (1999). their dynamic reorganization: a hypothesis for three deacetylase. Nature 403, 795–800 (2000). 27. Martin, S. G., Laroche, T., Suka, N., Grunstein, M. & distinctive features of cellular aging. Exp. Gerontol. 16. Kaeberlein, M., McVey, M. & Guarente, L. Gasser, S. M. Relocalization of telomeric Ku and SIR 33, 555–570 (1998). The SIR2/3/4 complex and SIR2 alone promote proteins in response to DNA strand breaks in yeast. 6. Goldstein, S. Replicative senescence: the human longevity in Saccharomyces cerevisiae by two different Cell 97, 621–633 (1999). fibroblast comes of age. Science 249, 1129–1133 mechanisms. Genes Dev. 13, 2570–2580 (1999). References 25 and 27 show that DNA damage (1990). 17. Straight, A. F. et al. Net1, a Sir2-associated nucleolar triggers the relocalization of the yeast Sir-silencing 7. Campisi, J. Senescent cells, tumor suppression, and protein required for rDNA silencing and nucleolar complex to sites of DNA breaks and causes nuclear organismal aging: good citizens, bad neighbors. Cell integrity. Cell 97, 245–256 (1999). changes that are reminiscent of normal ageing in 120, 513–522 (2005). 18. Shou, W. et al. Exit from mitosis is triggered by Tem1- yeast. 8. Kennedy, B. K. et al. Redistribution of silencing dependent release of the protein phosphatase Cdc14 28. Tamburini, B. A. & Tyler, J. K. Localized histone proteins from telomeres to the nucleolus is associated from nucleolar RENT complex. Cell 97, 233–244 acetylation and deacetylation triggered by the with extension of life span in S. cerevisiae. Cell 89, (1999). homologous recombination pathway of double-strand 381–391 (1997). 19. Visintin, R. et al. The phosphatase Cdc14 triggers DNA repair. Mol. Cell. Biol. 25, 4903–4913 (2005). 9. Sinclair, D. A. & Guarente, L. Extrachromosomal rDNA mitotic exit by reversal of Cdk-dependent 29. McMurray, M. A. & Gottschling, D. E. An age-induced circles — a cause of aging in yeast. Cell 91, phosphorylation. Mol. Cell 2, 709–718 (1998). switch to a hyper-recombinational state. Science 301, 1033–1042 (1997). 20. Kennedy, B. K., Austriaco, N. R., Jr, Zhang, J. & 1908–1911 (2003). 10. Chakalova, L., Debrand, E., Mitchell, J. A., Guarente, L. Mutation in the silencing gene SIR4 can 30. Eriksson, M. et al. Recurrent de novo point mutations Osborne, C. S. & Fraser, P. Replication and delay aging in S. cerevisiae. Cell 80, 485–496 in lamin A cause Hutchinson–Gilford progeria transcription: shaping the landscape of the genome. (1995). syndrome. Nature 423, 293–298 (2003). Nature Rev. Genet. 6, 669–677 (2005). 21. Watt, P. M., Louis, E. J., Borts, R. H. & Hickson, I. D. 31. Scaffidi, P. & Misteli, T. Reversal of the cellular 11. Hennekam, R. C. Hutchinson–Gilford progeria Sgs1: a eukaryotic homolog of E. coli RecQ that phenotype in the premature aging disease syndrome: review of the phenotype. Am. J. Med. interacts with topoisomerase II in vivo and is required Hutchinson–Gilford progeria syndrome. Nature Med. Genet. A 140, 2603–2624 (2006). for faithful chromosome segregation. Cell 81, 11, 440–445 (2005). 12. Narita, M. et al. Rb‑mediated heterochromatin 253–260 (1995). 32. Haithcock, E. et al. Age-related changes of nuclear formation and silencing of E2F target genes during 22. Yu, C. E. et al. Positional cloning of the Werner’s architecture in Caenorhabditis elegans. Proc. Natl cellular senescence. Cell 113, 703–716 (2003). syndrome gene. Science 272, 258–262 (1996). Acad. Sci. USA 102, 16690–16695 (2005). Describes how cellular senescence leads to the 23. Sinclair, D. A., Mills, K. & Guarente, L. Accelerated 33. Shiloh, Y. Ataxia-telangiectasia: closer to unraveling formation of facultative heterochromatic foci, which aging and nucleolar fragmentation in yeast sgs1 the mystery. Eur. J. Hum. Genet. 3, 116–138 (1995). alter the nuclear architecture. Alterations in mutants. Science 277, 1313–1316 (1997). 34. Smilenov, L. B. et al. Influence of ATM function on nuclear architecture change the expression of cell- 24. McAinsh, A. D., Scott-Drew, S., Murray, J. A. & telomere metabolism. Oncogene 15, 2659–2665 cycle regulators and can cause cell-cycle arrest. Jackson, S. P. DNA damage triggers disruption of (1997).

REVIEWS

35. Verdun, R. E. & Karlseder, J. The DNA damage 54. Pruitt, K. et al. Inhibition of SIRT1 reactivates silenced 72. Raghavan, S. C. & Lieber, M. R. DNA structures at machinery and homologous recombination pathway cancer genes without loss of promoter DNA chromosomal translocation sites. Bioessays 28, act consecutively to protect human telomeres. Cell hypermethylation. PLoS Genet. 2, e40 (2006). 480–494 (2006). 127, 709–720 (2006). 55. Bordone, L. et al. Sirt1 regulates insulin secretion by 73. Welle, S., Brooks, A. I., Delehanty, J. M., Needler, N. & 36. Greenwell, P. W. et al. TEL1, a gene involved in repressing UCP2 in pancreatic b cells. PLoS Biol. 4, Thornton, C. A. Gene expression profile of aging in controlling telomere length in S. cerevisiae, is e31 (2006). human muscle. Physiol. Genomics 14, 149–159 homologous to the human ataxia telangiectasia gene. 56. Picard, F. et al. Sirt1 promotes fat mobilization in (2003). Cell 82, 823–829 (1995). white adipocytes by repressing PPAR-γ. Nature 429, 74. Harman, D. Aging: a theory based on free radical and 37. Gaubatz, J. W. & Cutler, R. G. Mouse satellite DNA is 771–776 (2004). radiation chemistry. J. Gerontol. 11, 298–300 (1956). transcribed in senescent cardiac muscle. J. Biol. Chem. 57. Yu, B. P. & Chung, H. Y. Adaptive mechanisms to 75. Loden, M. & van Steensel, B. Whole-genome views of 265, 17753–17758 (1990). oxidative stress during aging. Mech. Ageing Dev. 127, chromatin structure. Chromosome Res. 13, 289–298 38. Shen, S., Liu, A., Li, J., Wolubah, C. & Casaccia- 436–443 (2006). (2005). Bonnefil, P. Epigenetic memory loss in aging 58. Liu, B. et al. Genomic instability in laminopathy- 76. Kouzarides, T. Chromatin modifications and their oligodendrocytes in the corpus callosum. Neurobiol. based premature aging. Nature Med. 11, 780–785 function. Cell 128, 693–705 (2007). Aging 19 December 2006 (doi:10.1016/ (2005). 77. Craig, J. M. Heterochromatin — many flavours, j.neurobiolaging.2006.10.026). 59. Csoka, A. B. et al. Genome-scale expression profiling common themes. Bioessays 27, 17–28 (2005). 39. Imai, S. et al. Dissociation of Oct-1 from the nuclear of Hutchinson–Gilford progeria syndrome reveals 78. Dernburg, A. F. et al. Perturbation of nuclear peripheral structure induces the cellular aging- widespread transcriptional misregulation leading to architecture by long-distance chromosome associated collagenase gene expression. Mol. Biol. mesodermal/mesenchymal defects and accelerated interactions. Cell 85, 745–759 (1996). Cell 8, 2407–2419 (1997). atherosclerosis. Aging Cell 3, 235–243 (2004). 79. Kovtun, I. V. et al. OGG1 initiates age-dependent CAG 40. Zhang, R. et al. Formation of macroH2A-containing 60. Niedernhofer, L. J. et al. A new progeroid syndrome trinucleotide expansion in somatic cells. Nature 447, senescence-associated heterochromatin foci and reveals that genotoxic stress suppresses the 447–452 (2007). senescence driven by ASF1a and HIRA. Dev. Cell 8, somatotroph axis. Nature 444, 1038–1043 (2006). 80. Bokov, A., Chaudhuri, A. & Richardson, A. The role of 19–30 (2005). A mouse model for a human nucleotide-excision oxidative damage and stress in aging. Mech. Ageing 41. Dellaire, G. & Bazett-Jones, D. P. PML nuclear bodies: repair defect shows premature ageing and gene- Dev. 125, 811–826 (2004). dynamic sensors of DNA damage and cellular stress. expression changes similar to those observed 81. Schriner, S. E. et al. Extension of murine life span by Bioessays 26, 963–977 (2004). during normal ageing. overexpression of catalase targeted to mitochondria. 42. Herbig, U., Ferreira, M., Condel, L., Carey, D. & 61. van Attikum, H. & Gasser, S. M. The histone code at Science 308, 1909–1911 (2005). Sedivy, J. M. Cellular senescence in aging primates. DNA breaks: a guide to repair? Nature Rev. Mol. Cell 82. Mostoslavsky, R. et al. Genomic instability and aging- Science 311, 1257 (2006). Biol. 6, 757–765 (2005). like phenotype in the absence of mammalian SIRT6. 43. Lu, T. et al. Gene regulation and DNA damage in the 62. Downs, J. A. et al. Binding of chromatin-modifying Cell 124, 315–329 (2006). ageing human brain. Nature 429, 883–891 (2004). activities to phosphorylated histone H2A at DNA 83. Lombard, D. B. et al. DNA repair, genome stability, Gene-expression profiling reveals DNA-damage- damage sites. Mol. Cell 16, 979–990 (2004). and aging. Cell 120, 497–512 (2005). induced global gene repression in the ageing brain. 63. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. & 84. Van Remmen, H. et al. Life-long reduction in MnSOD 44. Lee, C. K., Weindruch, R. & Prolla, T. A. Gene- Chen, D. J. ATM phosphorylates histone H2AX in activity results in increased DNA damage and higher expression profile of the ageing brain in mice. Nature response to DNA double-strand breaks. J. Biol. Chem. incidence of cancer but does not accelerate aging. Genet. 25, 294–297 (2000). 276, 42462–42467 (2001). Physiol. Genomics 16, 29–37 (2003). 45. Lee, C. K., Klopp, R. G., Weindruch, R. & Prolla, T. A. 64. Ward, I. M. & Chen, J. Histone H2AX is 85. Lund, J. et al. Transcriptional profile of aging in Gene expression profile of aging and its retardation by phosphorylated in an ATR-dependent manner in C. elegans. Curr. Biol. 12, 1566–1573 (2002). caloric restriction. Science 285, 1390–1393 (1999). response to replicational stress. J. Biol. Chem. 276, 86. Hoppe, G. J. et al. Steps in assembly of silent 46. Kayo, T., Allison, D. B., Weindruch, R. & Prolla, T. A. 47759–47762 (2001). chromatin in yeast: Sir3-independent binding of a Influences of aging and caloric restriction on the 65. Rogakou, E. P., Boon, C., Redon, C. & Bonner, W. M. Sir2/Sir4 complex to silencers and role for Sir2- transcriptional profile of skeletal muscle from rhesus Megabase chromatin domains involved in DNA dependent deacetylation. Mol. Cell. Biol. 22, monkeys. Proc. Natl Acad. Sci. USA 98, 5093–5098 double-strand breaks in vivo. J. Cell Biol. 146, 4167–4180 (2002). (2001). 905–916 (1999). 47. Fraser, H. B., Khaitovich, P., Plotkin, J. B., Paabo, S. & 66. Sanders, S. L. et al. Methylation of histone H4 lysine Acknowledgements Eisen, M. B. Aging and gene expression in the primate 20 controls recruitment of Crb2 to sites of DNA We thank B. North for critical reading of the manuscript. The brain. PLoS Biol. 3, e274 (2005). damage. Cell 119, 603–614 (2004). Sinclair laboratory is supported by National Institutes of 48. Park, S. K. & Prolla, T. A. Gene expression profiling 67. Giannattasio, M., Lazzaro, F., Plevani, P. & Muzi- Health grants and the Paul F. Glenn Laboratories for the studies of aging in cardiac and skeletal muscles. Falconi, M. The DNA damage checkpoint response Biological Mechanisms of Aging. P.O. is supported by Cardiovasc. Res. 66, 205–212 (2005). requires histone H2B ubiquitination by Rad6–Bre1 the National Space Biomedical Research Institute. 49. Bahar, R. et al. Increased cell-to-cell variation in gene and H3 methylation by Dot1. J. Biol. Chem. 280, expression in ageing mouse heart. Nature 441, 9879–9886 (2005). Competing interests statement 1011–1014 (2006). 68. Kim, S., Benguria, A., Lai, C. Y. & Jazwinski, S. M. The authors declare competing financial interests. Gene-expression patterns vary between individual Modulation of life-span by histone deacetylase genes cells of aged cardiomyocytes. These changes in Saccharomyces cerevisiae. Mol. Biol. Cell 10, appear to be stochastic and are caused by DNA 3125–3136 (1999). DATABASES damage. 69. Huyen, Y. et al. Methylated lysine 79 of histone H3 Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. 50. Pletcher, S. D. et al. Genome-wide transcript profiles targets 53BP1 to DNA double-strand breaks. Nature fcgi?db=gene in aging and calorically restricted Drosophila 432, 406–411 (2004). ATM | LMNA | WRN melanogaster. Curr. Biol. 12, 712–723 (2002). The crystal structure of a methyl-binding DNA OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. 51. Lin, S. J., Defossez, P. A. & Guarente, L. Requirement repair factor reveals an evolutionarily conserved fcgi?db=OMIM of NAD and SIR2 for life-span extension by calorie role for histone methylation in mammalian DNA ataxia telangiectasia | Hutchinson–Gilford progeria restriction in Saccharomyces cerevisiae. Science 289, repair. syndrome | Werner syndrome 2126–2128 (2000). 70. Botuyan, M. V. et al. Structural basis for the UniProtKB: http://ca.expasy.org/sprot 52. Rogina, B. & Helfand, S. L. Sir2 mediates longevity in methylation state-specific recognition of histone 53BP1 | Cdc14 | H2AX | Net1 | Sir2 | Sir3 | Sir4 | SIRT1 | XPF the fly through a pathway related to calorie restriction. H4‑K20 by 53BP1 and Crb2 in DNA repair. Cell 127, Proc. Natl Acad. Sci. USA 101, 15998–16003 1361–1373 (2006). FURTHER INFORMATION (2004). 71. Malins, D. C. et al. Oxidative changes in the DNA of David A. Sinclair’s homepage: http://medapps.med.harvard. 53. Cohen, H. Y. et al. Calorie restriction promotes stroma and epithelium from the female breast: edu/agingresearch/pages/faculty.htm mammalian cell survival by inducing the SIRT1 potential implications for breast cancer. Cell Cycle 5, All links are active in the online pdf. deacetylase. Science 305, 390–392 (2004). 1629–1632 (2006).