The emerging role of epigenetics in cellular and organismal aging

The emerging role of epigenetics in cellular and organismal aging

Experimental Gerontology 38 (2003) 1299–1307 www.elsevier.com/locate/expgero The emerging role of epigenetics in cellular and organismal aging Debdut...

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Experimental Gerontology 38 (2003) 1299–1307 www.elsevier.com/locate/expgero

The emerging role of epigenetics in cellular and organismal aging Debdutta Bandyopadhyay, Estela E. Medrano* Huffington Center on Aging and Departments of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, One Baylor Plaza M320, Houston, TX 77030, USA

Abstract Genome modifications resulting from epigenetic changes appear to play a critical role in the development and/or progression of cancer. Scatter experimental evidence suggests that epigenetic changes could also be critical determinants of cellular senescence and organismal aging. Here we review the current evidence and discuss how imbalances in chromatin remodelers might trigger irreversible growth arrest in proliferating cells and tissues. Experimental data using drugs that target specific chromatin remodeling enzymes suggest that such approach could lead to the development of novel therapeutic modalities for the prevention or amelioration of some age-related dysfunctions. q 2003 Elsevier Inc. All rights reserved. Keywords: Chromatin; Chromatin modifiers; DNA-methylation; Histone methylation; Histone acetylation

1. Introduction The nucleotide sequence in genomic DNA is not the only genetic information in the cell. Epigenetics, which means outside genetics, is the study of stable alterations in gene expression that arise during development and cellular proliferation, and, importantly, by the influence of the environment (reviewed in Jaenisch and Bird, 2003; Sutherland and Costa, 2003). Epigenetic modifications consist of DNA-methylation, histone post-transcriptional modifications including methylation, acetylation and phosphorylation, and ATP-dependent, structural modifications of chromatin. The characterization and functional analysis of epigenetic modifications involved in tumorigenesis has been the focus of intense research throughout the last decade (reviewed in Neumeister et al., 2002). Also, more than two decades ago, it was proposed that ‘aging of proliferating cells’ results from genome reorganization occurring during the division cycle (Macieira-Coelho, 1980). However, progress in the areas of epigenetics and cellular and organismal aging has been slow and sporadic. Several reasons could coalesce for such paucity, being the complexity of aging perhaps the most important, but also lack of specific reagents and ill-defined targets. However, the recent discovery that overexpression of Sir2, a NADþ-dependent histone deacetylase extends yeast and worm lifespan * Corresponding author. Tel.: þ 1-713-798-1569; fax: þ1-713-798-4161. E-mail address: [email protected] (E.E. Medrano). 0531-5565/$ - see front matter q 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.09.009

(Hekimi and Guarente, 2003) has triggered a renewed interest in the possible role of chromatin remodeling in aging and replicative senescence. DNA replication and transcription are coupled to cellular proliferation. Thus, the corresponding machineries need to tether to and stroll through chromatin. In proliferating cells, chromatin is mostly found in a ‘permissive’ or euchromatic state, at least, in loci associated with active transcription of growthregulatory genes. Conversely, it has been proposed that reassembly of repressive chromatin domains (heterochromatin) may contribute to senescence (Howard, 1996). In this brief review, we discuss how different mechanisms of chromatin remodeling may play a critical role in cellular and organismal aging.

2. Mechanisms involved in chromatin remodeling 2.1. DNA-methylation CpG island-methylation is associated with stable gene silencing and formation of heterochromatin structures (Eden et al., 1998; Jones and Laird, 1999). Transcriptional repressors, including MeCP2 and methyl-CpG-binding proteins (MBD1 – MBD4) (Billard et al., 2002; El Osta et al., 2002; Rietveld et al., 2002) specifically bind hypermethylated DNA segments in association with histone deacetylases (HDACs) (Feng and Zhang, 2001; Jones et al., 1998; Nan et al., 1998; Ng et al., 1999).

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Two decades ago, it was proposed that gradual loss of DNA-methylation could function as a ‘counting mechanism’ for senescence (Hoal-van Helden and van Helden, 1989; Wilson and Jones, 1983). CpG methylation decreases with increased population doublings of normal cells in culture (Wilson and Jones, 1983) (Fairweather et al., 1987) and during organismal aging (Hornsby et al., 1992; Singhal et al., 1987). Consistent with loss of DNA-methylation, the activity of DNA-methyl transferase (the enzyme, that catalyses the methylation of CpG island) also decreases with increasing population doubling in normal human fibroblasts (Vertino et al., 1994). Supporting the DNAmethylation ‘counting hypothesis’, most immortal cell lines maintain constant levels of genomic DNA-methylation (Young and Smith, 2001). How can we reconcile the idea that DNA-methylation represses transcription with the notion that DNA-methylation decreases during senescence? Methylation may repress a set of growth inhibitory genes the repression of which could become less stringent with increased population doublings (Baylin and Herman, 2000; Jones and Baylin, 2002). This hypothesis is supported by findings demonstrating that inhibition of DNA-methyltransferase (DNMT) results in the transcriptional activation of the p21Waf-1 gene in normal human fibroblasts (Young and Smith, 2001). Paradoxically, CpG island-methylation progressively increases with age at multiple gene loci in normal colorectal epithelium, suggesting genome-wide molecular alterations with potential to silence gene expression (Issa, 2002). However, there was considerable variation in the degree of methylation among individuals of comparable ages. Such variation could be related to genetic factors, lifestyle, or environmental exposures. Thus, it will be important to determine whether changes in DNA-methylation are stochastic (Jaenisch and Bird, 2003), or somewhat ‘programmed’ as animals age, and whether the environment and diet also contribute to such changes. It is expected that such studies will present substantial difficulties due to

the extreme complexity and unpredictability of DNAmethylation outcomes. DNA hypermethylation is associated with silencing of tumor suppressor genes, and thus with oncogenesis. Surprisingly, hypomorphic DNMT1 alleles result in the development of aggressive T cell lymphomas that display a high frequency of chromosome 15 trisomy (Gaudet et al., 2003). Thus, DNA hypomethylation can also play a causal role in tumor formation, possibly by promoting chromosomal instability (reviewed in Lengauer, 2003). 2.2. Histone modifications Covalent modification of N-terminal tails of histones H3 and H4 also contribute to chromatin remodeling. These modifications include acetylation, methylation, phosphorylation, and ubiquitination. The transcriptional status of promoters correlates with specific modifications of core histones. These findings led to the concept of a ‘Histone Code’ that describes how different combinations of histone modifications may lead either to activation or silencing of gene transcription (reviewed in Jenuwein and Allis, 2001). According to this theory, specific modifications of histones H3 and H4 tails correlate with either transcriptional activation or repression. Histone H3 has four potential modification sites: Lys-4 (methylation), Lys-9 (acetylation/ methylation), Ser-10 (phosphorylation) Lys-14 (acetylation) and Arg-17 (methylation). An active transcriptional state is associated with phosphorylation of Ser-10, which in turn facilitates acetylation of Lys-14, as well as methylation of Lys-4 (Cheung et al., 2000; Lo et al., 2000) (Fig. 1). Ser-10 phosphorylation also appears to facilitate acetylation of Lys-9, which in turn, prevent methylation of Lys-9 of H3 (Rea et al., 2000). In addition, methylation of Arg-3 in H4, that precedes acetylation of Lys-8 and -12, correlates with transcriptionally active chromatin (Wang et al., 2001). Conversely, deacetylation of Lys-9 and -14, dephosphorylation of Ser-10, and methylation of Lys-9 correlate with a repressive transcriptional state (Fig. 1). Thus,

Fig. 1. Modification of different amino acid residues in histone H3 leads either to activation or repression of transcription.

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post-translational modifications of histone tails are under tight regulation to maintain cellular homeostasis. 2.2.1. Acetylation/deacetylation of histones and cellular senescence The histone acetyl-transferase p300 HAT regulates genome-wide activities through histone acetylation and is a powerful protein acetyl-transferase as well (Chan and La Thangue, 2001). p300 HAT is also a molecular integrator that controls transcription and function of cell cycle regulatory proteins, differentiation, DNA repair (Campisi, 2001; Ogryzko et al., 1996) and apoptosis (Howard, 1996; Landry et al., 2000). p300 and CBP affect transcription in various ways: (1) they promote access of transcription factors to DNA (Lee et al., 1993) by neutralizing the positive charge of histones (Turner, 1991); (2) they destabilize higher order chromatin structure (Luger et al., 1997; Tse et al., 1998); and (3) they promote the processivity of RNA polymerase (Ura et al., 1997). In addition, p300 and CBP can function as protein acetyltransferases. Targets include the E2F family of transcription factors (Martinez-Balbas et al., 2000; Marzio et al., 2000), the tumor suppressor p53 (Liu et al., 1999), and the Myb protein (Tomita et al., 2000). In general, acetylation enhances the DNA-binding activity of the target proteins. Histone deacetylation also plays a key role in transcriptional regulation. There are two families of proteins with HDAC activity: the classical HDAC family and the SIR2 family of NADþ-dependent HDACs. The classical HDACs can be sub-categorized into two phylogenetic classes, Class I HDACs (HDAC1, 2, 3 and 8) and class II HDACs (HDAC4, 5, 6, 7, 9, and 10). HDACs deacetylate the epsilon amino group of lysyl-side residues, resulting in non-permissive, compact heterochromatin structures. HDACs are recruited to DNA through DNAbinding proteins. For example, RB –E2F complexes containing HDACs (Luo et al., 1998; Magnaghi-Jaulin et al., 1998) function as transcriptional repressors. As mentioned previously, HDACs can associate with CpG-binding proteins including MBD2. HDAC1 was also identified in a tetrameric complex that includes Max, Mad1, and Sin3B. Mad1-mediated cell growth arrest could be enhanced by the presence of Sin3B and HDAC1 in the complexes (Sommer et al., 1997). 2.2.1.1. The Sir2 family of histone deacetylases. We will discuss briefly Sir2 activity and its role in aging since there are excellent reviews on this subject (Chang and Min, 2002; Hekimi and Guarente, 2003; Lin and Guarente, 2003; Sinclair, 2002). The silent information regulator (SIR) family of proteins is involved in multiple and important cellular events including transcriptional silencing, chromatin remodeling, mitosis and lifespan duration (Guarente, 2000). Sir2-like enzymes catalyze a reaction in which the cleavage of NAD(þ ) and histone and/or protein deacetylation is coupled to the formation of O-acetyl-ADP-ribose.

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The dependence of the reaction on both NAD(þ ) and the generation of this potential second messenger offer new clues to understanding the function and regulation of nuclear, cytoplasmic and mitochondrial Sir2-like enzymes. The yeast ySir2 is involved in gene silencing, chromosomal stability and aging (Denu, 2003). Sir2 can deacetylate histones H3 and H4, is required for silencing at telomere ends (Aparicio et al., 1991; Strahl-Bolsinger et al., 1997), and for repressing rDNA (Fritze et al., 1997). In budding yeast, deletion of SIR2 shortened the lifespan, whereas an extra copy of this gene increased life span, demonstrating the importance of Sir2 family in aging (Kaeberlein et al., 1999). The NAD-dependent HDAC activity of this family of proteins is broadly conserved, and is thought to be important for their role as chromatin silencer (Imai et al., 2000). Since NAD is one of the critical molecules of metabolic pathway and gene silencing is one of the key points of cellular aging, it was very provocative to search if Sir2 family of proteins is involved in caloric restriction mediated elongation of life span. It was suggested that under restricted caloric condition, carbon flow in the glycolysis and TCA cycle is much lower and less NAD may be siphoned from the common pool, allowing more Sir2 to bind to it. Thus, the Sir2 proteins may link metabolic rate to the pace of aging by sensing the NAD levels and generating the mandated level of chromatin silencing (Guarente, 2000). Recent studies have demonstrated that human SIRT1, a Sir2 homologue, functions as a p53 deacetylase. Deacetylation of p53 by SIRT1, which impairs p53-transcriptional activity (Vaziri et al., 2001) prevents cellular senescence and apoptosis induced by DNA damage and stress. In addition, SIRT2, also a human homologue of yeast Sir2, is involved in mitosis in human cells and its overexpression leads to prolongation of the mitotic phase (Dryden et al., 2003). More studies are needed to determine what functions of the SIR proteins are strictly necessary for its lifespan extension activity. Intriguingly, embryonic stem cells null for sir2a do not have elevated levels of acetylated histones and do not ectopically express silent genes (McBurney et al., 2003). These results suggest that either mammalian sir2a has different substrates compared to yeast sir2, or that other mammalian sir proteins may be more closely related in function to yeast sir2. 2.2.1.2. p300 and HDAC1: paradigms and controversies. The CBP2/2 and p3002/2 embryos die between days 9 and 11.5. Their main defects are severe developmental retardation, reduced size, failed neural tube closure and altered cardiac ventricular trabeculation (Blobel, 2000). Importantly, p300 deficient fibroblasts show slow proliferation and rapid senescence, suggesting that p300 is involved in maintaining the proliferative state of cells (Yao et al., 1998). Supporting this hypothesis, more recent data shows that p300/CBP is required for the G1/S transition (Ait-Si-Ali et al., 2000; Lee et al., 1993). In addition, the levels of p300 and CBP decrease with increasing population doublings of

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human melanocytes (Bandyopadhyay et al., 2002). In these cells, p300 HAT appears to be essential for proliferation, as overexpression of a dominant negative p300 HAT protein (DN p300 HAT), or treatment with Lys-CoA, a specific chemical inhibitor of p300 HAT, triggers early senescence, as measured by irreversible loss of cell division and SA-bgal staining (Bandyopadhyay et al., 2002). This suggests that sustained levels of histone H3 and H4 acetylation may be required for lifespan extension of cultured cells. This hypothesis is supported by recent studies demonstrating that feeding Drosophila with 4-phenylbutyrate (PBA), a HDAC inhibitor, results in substantial extension of its lifespan (Kang et al., 2002). In addition, expression of a hypomorphic (partial loss-of-function) rpd3 deacetylase allele increases Drosophila longevity (Rogina et al., 2002) whereas greatly reduced levels of this deacetylase result in lethality (Mannervik and Levine, 1999). Although there is little information regarding levels and activity of HATs in old mammals, two studies have demonstrated a decline in p300/CBP levels in tissues of old mice and rats. The HAT activity of CBP/p300 is attenuated in the liver, muscle and testes of aged mice (Li et al., 2002), whereas p300 levels decline in motoneurons of the spinal nucleus of the bulbocavernosus in rats (Matsumoto, 2002). Considering that p300 functions as a coregulator of multiple transcription factors (Chan and La Thangue, 2001) reduction of its levels can also have deleterious effects for tissue-specific gene activity in nonproliferating cells. There are, however, contradictory results that need to be discussed. Based on the data showing that p300 is required for fibroblast and melanocyte proliferation, one could predict that inhibition of HDACs should favor lifespan extension. However, sodium butyrate (NaB), an HDACinhibitor, also induces a senescent-like phenotype in human fibroblasts (Ogryzko et al., 1996) and melanoma cells (Bandyopadhyay and Medrano, unpublished). This state resembles replicative senescence in terms of morphology, saturation density and cell cycle distribution, including accumulation of hypophosphorylated RB and induction of the cyclin-dependent kinase inhibitor p21Waf-1 (Xiao et al., 1997). Interestingly, NaB-treated cells display high levels of histone acetylation compared to proliferating cells, suggesting that inhibition of HDAC1 results in derepression of growth inhibitory genes including p21. Thus, how can these apparently paradoxical results be reconciled? A likely explanation could be the necessity of the cells to maintain a critical balance between acetylated and deacetylated chromatin domains, in order to sustain proliferation. Whereas hyperacetylation caused by loss of HDAC activity could induce antiproliferative genes in chromatin regions (Howard, 1996), loss of acetylation may shift the balance towards repressive heterochromatin causing silencing of genes associated with cell cycle progression. This hypothesis is supported by the observation that targeted disruption of both HDAC1 alleles results in

embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, decreased cyclin-associated kinase activity, and elevated levels of p21 and p27. Importantly, the expression of HDAC2 and HDAC3 is induced in HDAC1-defficient cells, but cannot compensate for its loss, suggesting a unique function of HDAC1 (Lagger et al., 2002). 2.2.2. Histone methylation Methylation targets lysine and arginine residues in histone tails. Methylation of lysine-4 and arginine-17 in Histone H3 correlates with active gene expression whereas lysine-9 methylation leads to silencing (Boggs et al., 2002; Bauer et al., 2002; Lachner et al., 2001), and methylation of arginine-8 in histone H4 facilitates transcription activation by nuclear hormone receptor (Wang et al., 2001). Evidence for the involvement of histone methylation in establishing a heterochromatin state comes from the finding that MeK9 in H3 (MeK9-H3) is a binding site for the heterochromatin binding protein 1 (HP1) (Bannister et al., 2001; Lachner et al., 2001). The histone methyltransferase Suv39H1 was also found to interact and colocalize with HP1 (Lachner et al., 2001). Furthermore, the DNA methyltransferase, DNMT1 also co-localized with HP1 at heterochromatic sites in embryonic stem cells (Bachman et al., 2001). DNMT1 and DNMT3a associate with SUV39H1 and HP1 (Fuks et al., 2003) indicating a strong interplay between histone methylation and DNA-methylation. Such multiprotein complexes may be required to achieve high levels of silencing specificity at targeted promoters. Repression by HP1 and Suv39H1 at certain loci appear to require the retinoblastoma protein RB (Nielsen et al., 2001). RB is necessary for the binding of HP1 to the cyclin E promoter, suggesting that Suv39H1 – HP1 complex has a role in repression of euchromatic genes. Consistent with these results, it has been proposed that the decision to enter senescence, is partially determined by a histone methyltransferase that, together with RB, HP1 and SUV39H1, alters the chromatin structure and silence E2F target genes (Narita et al., 2003). Supporting a possible role of histone methylation in aging, trimethylation of lysine-20 in Histone H4 increases in an age-dependent manner in rat livers (Sarg et al., 2002). It remains to be determined, whether, such modification is a consequence of maturation and aging of tissues and organs, or whether it can have a causative effect in aging. A summary of how histone modifications can lead to either growth arrest or proliferation is shown in Fig. 2. 2.3. ATP-dependent chromatin remodeling: the SWI/SNF complexes Other chromatin remodeling mechanisms utilize the energy of ATP-hydrolysis to modify chromatin structure. ATP-dependent chromatin remodeling complexes contain

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Fig. 2. A simplified scheme showing that similar type of modification may exert opposite effect on cellular growth, depending on the subset of gene(s) being modified. For example, promoter acetylation of growth promoting genes may lead to cellular proliferation, whereas acetylation of growth inhibitory gene promoters can result in their re-expression and consequent cell cycle arrest.

an ATPase subunit that belongs to the SNF2 superfamily of proteins. Based on the identity of the subunits, they are classified into two main groups, the SWI2/SNF2 and the SWI (ISWI) groups (Eisen et al., 1995). Members of the SWI/SNF group of proteins include yeast Swi2/Snf2, yeast RSC, Drosophila Brahma complex and the human Brg1 and Brm1 proteins. These proteins contain a highly conserved ATPase domain and a C-terminal bromodomain (Vignali et al., 2000). The second group of ATP-dependent chromatin remodelers contains the ISWI protein as the ATPase. A group of proteins with similar functions were purified from human cells and named the Mi-2 family of proteins. The members include NURD, NuRD, NRD (Tong et al., 1998; Xue et al., 1998; Zhang et al., 1998). The precise mechanism by which these complexes alter the chromatin structure is not completely understood

yet. Current models suggest an initial step required for binding of the complex to DNA. Upon ATP-addition, the histone –DNA interaction is altered, leading to loosening of chromatin structure. The final remodeling step may result in transferring the histone octamer to different DNA segment in trans or in sliding of the octamer in the same DNA-fragment in cis. The sequence step of this type of remodeling is thought to be dependent on the nucleosome context at a given promoter, and can lead to either activation or repression of transcription (Vignali et al., 2000). 2.3.1. Role of SWI/SNF complexes in cell growth regulation Paradoxical data suggests that the activity of ATPdependent chromatin remodelers is very complex. hBRG1 appears to be necessary for normal mitotic growth and

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transcription (Khavari et al., 1993). Similarly, Sfh 1p, a component of yeast RSC complex is also shown to be required for cell cycle progression. Transient transfection studies demonstrated that SWI/SNF complexes are involved in transcriptional activation of some members of the nuclear receptor superfamily, including the glucocorticoid receptor (Chiba et al., 1994; Muchardt and Yaniv, 1993; Singh et al., 1995). Also, BRG1 interacts with b-catenin, an oncogenic protein, to promote transcriptional activity of Tcf responsive reporter genes (Barker et al., 2001). Conversely, substantial experimental evidence suggests that BRG1 and the highly homologous protein Brm1 function as transcriptional repressors and growth inhibitors. BRM knockout mice show increased cell proliferation in the liver (Reyes et al., 1998). In addition, RB and hBrm are required to repress E2F-mediated transcriptional activation (Trouche et al., 1997), and, in a concerted manner, HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF complexes, repress cyclin E and cyclin A, respectively (Zhang et al., 2000). Evidence involving SWI/SNF complexes in aging is limited but suggestive. BRG1 is capable of inducing a senescent-like morphology in the human adrenal cortex carcinoma derived cell line SW13 (Shanahan et al., 1999). These cells do not divide and express markers indicative of replicative senescence, including, SA b-galactosidase activity. Interestingly, cyclin E and cyclin D1 can abrogate BRG1 activity, reducing the number of ‘senescent-like’ cells. A caveat of such studies is the use of tumor cells only. In vivo, Brm1 complexes appear to regulate aging in the liver. Brm1, but not BRG1, levels increase in the liver of old mice and correlate with the appearance of age-specific repressive complexes containing Brm1, C/EBPa, a liver transcription factor, RB and E2F4 (Iakova et al., 2003). Increased levels of Brm are crucial for the formation of a repressive complex, as incubation of nuclear proteins from young animals with an excess of Brm1 leads to the appearance of a complex similar to those found in old livers. Since Brm1 is usually a component of SWI/SNF multi-protein complexes, it might be informative to determine whether other components of these complexes are present in the Brm1/C/EBPa/RB/E2F4 complex found in the aging liver. Similarly, it will be interesting to determine whether Brm1 levels also increase in non-dividing, post-mitotic cells in aged mice.

3. Conclusions It has been proposed that chromatin could be a useful tool for the study of genome functions in cancer (Urnov, 2003). Scattered data suggests that chromatin could also regulate genome changes in aging. Transcriptional ‘fingerprints’ unique to cellular senescence (Zhang et al., 2003) suggest the conversion of specific heterochromatin domains to euchromatin. Equally possible, permanently relocating cell

growth promoting genes to the heterochromatic compartment could result in cellular senescence (Bandyopadhyay et al., 2002). Together, these results indicate that sustained proliferation entails a critical balance of chromatin remodelers; and increase or decrease of such molecules can profoundly alter genome structure. It has been proposed that defects in genome stability could lead to aging in humans and mice (Hasty et al., 2003). Chromatin organization and remodeling genes including histones and HDACs of the SIR2 and HDAC (Rpd3) family contribute to genomic stability in somatic cells of C. elegans (Pothof et al., 2003). Similarly, chromatin remodelers are associated with damage recovery in Saccharomyces cerevisiae (Begley et al., 2002). New technologies such as RNA interference (RNAi) could be used in C. elegans and S. cerevisiae to test the role of individual and combinations of chromatin remodelers in chromosome stability and lifespan duration. Similar studies in mammalian cells may provide the groundwork for understanding the molecular bases for the still mysterious link between aging and cancer. Finally, unlike stable genome alterations, it is also possible to alter gene expression by specific drugs. HDAC inhibitors and demethylating agents are currently being tested as novel cancer therapies. Lifespan extension of Drosophila by feeding a HDAC inhibitor (PBA) suggests that the ‘fountain of youth’ might be within our own chromosomes. For the moment, it might be possible to design drugs that target specific chromatin modifications to improve the outcome of some age-related diseases.

Acknowledgements Space limitations have prevented us to reference a large number of key original papers and reviews. Work in the authors’ laboratory is supported by grants P01 AG19254 and R01 CA84282 from the National Institutes of Health.

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