We investigated the effects of age on cytosine methylation throughout the genome using a genome-wide assay of a type not previously exploited in the study of aging metabolically-active tissues such as liver and visceral fat. This study represents one of the first such genome-wide studies of the aging epigenome, and is the first to demonstrate not only genome-wide but also locus-specific differences in cytosine methylation in both liver and adipose tissue. We find that normal aging in genetically identical rats exposed to the same environment throughout life causes consistent tissue-specific dysregulation of cytosine methylation, and that these changes skew globally towards hypermethylation of unique sequences in the liver. This increased methylation is accompanied by hypomethylation at a smaller set of loci, and less pronounced effects in perinephric visceral fat. The epigenomic dysregulation appears to be non-random in terms of genomic sequence context, preferentially affecting specific loci and genomic compartments such as intergenic and conserved sequences. These epigenetic changes may be adaptive and secondary to other alterations in cellular physiology, in which case they represent potential biomarkers of the aging process and indicators of the heterogeneity of the pathophysiology of aging between tissues. However, as many of these changes occur in proximity to genes with well-established roles in metabolism and metabolic dysregulation, epigenomic dysregulation is a clear candidate for being a primary mediator of the pathogenesis of age-related metabolic disease.
The extremely limited overlap that we observe between liver- and fat-specific epigenetic dysregulation is fundamentally due to the limited epigenetic variability observed in fat with age (, ), and results in an extremely small number of loci that are dysregulated in both tissues with age. Identification of loci that are jointly dysregulated in different tissues with age may suggest a common age-response mechanism operating in both tissues, but our results do little to shed light on such a mechanism with such a small number of loci involved and the high false-positive rate in adipose tissue in the current study. Furthermore, the preponderance of unique changes in each tissue suggests that a common mechanism might explain only a part of the story. Instead, we hypothesize that varying tissue environments with age, as well as varying mitotic activity and cellular susceptibilities to accumulated damage, are the hallmarks of tissue-specific epigenomic dysregulation with age.
Why liver as opposed to fat should be subject to large epigenomic changes (, ) is somewhat counterintuitive, given that visceral fat is centrally involved in the pathogenesis of age-related diseases (Muzumdar et al. 2008
), and our prior expectation was that adipose tissue would have greater potential for epigenetic dysregulation due to the proximity of the nuclear DNA in adipocytes to free fatty acid flux and the accumulation of tissue macrophages, a phenomenon typical of aging (Einstein et al. 2008
). However, the tissue-specific epigenetic differences may be related to the distinct cell proliferation properties of the tissues, analogous to the distinct tissue-specific DNA mutational rates previously observed in brain and small intestine (Busuttil et al. 2007
). As liver is a relatively highly proliferative tissue type (Duncan et al. 2009
), it may be more susceptible than adipose tissue to the accumulation of mutations, not just those of DNA but also those of epigenetic organization, or ‘epimutations’. DNA replication involves the propagation of cytosine methylation patterns to daughter chromatids, with restoration of symmetrical methylation from an initially hemimethylated state by DNA methyltransferase 1 (DNMT1), thus preserving the pattern of methylation present in the parental cell. However, DNMT1 has measurable de novo
methylation activity and an estimated error rate of 0.3–5% (Vilkaitis et al. 2005
; Goyal et al. 2006
), thus epimutations are likely to occur with each cell division. A prior study has indicated that less mitotically-active cell types may be less prone to age-associated changes in cytosine methylation (Chu et al. 2007
), results concordant with our data. The age-related epigenomic dysregulation that arises in a non-dividing cell type such as mature adipocytes (Neese et al. 2002
) is more likely to reflect changes that occur within a single cell’s lifespan such as DNA repair-mediated loss of methylation (Barreto et al. 2007
; Meulle et al. 2008
While the locus-specific changes we observed are potentially valuable insights into the pathophysiology of aging, it is the striking tissue-specificity we observe that represents the most novel finding of our study. Because of these findings, we propose that epigenomic dysregulation in aging should be studied in a tissue-specific context, and that the lessons learned from one tissue cannot necessarily be applied to other tissues. The failure of a large-scale, quantitative study of cytosine methylation to find changes in DNA methylation with age (Eckhardt et al
. 2006a) may be due to their measurement of age-related differences as average values across the many loci and tissue types they studied, whereas our results indicate that a locus and cell type-specific approach to the same dataset may yield different results. The changes we observe in our study are unlikely to be due to a random pattern of loss of epigenetic regulation, as might be concluded from a study of age effects on the epigenomes of twins (Fraga et al. 2005
). Indeed, with an estimated false discovery rate of about 2.5% in liver and 21.3% in fat (δ=0.5), the epigenetic dysregulation we observe with age is likely due to reproducible, non-random biological differences.
The changes in methylation that we observed included but were not limited to promoter-proximal loci, making it difficult to predict whether these changes would have any consequences in terms of local transcription. We addressed this question by performing gene expression microarray studies on young and old rat livers, demonstrating a subset of loci at which changes were concordant. The role of intergenic loci in transcriptional regulation remains difficult to assess, but our data indicate that at least some such loci are potentially cis-reguatory and involved in cellular aging. Studies of the aging epigenome should not therefore be limited to promoter regions but should include other genomic contexts also.
We have to consider the possibility that fat- and more notably liver-specific dysregulation could be a product of a controlled tissue-specific response to accumulated stress with aging. This idea is supported by the results from our ontological and pathway analyses, which showed the enrichment of physiologically relevant loci in functional networks associated with metabolism and age-related diseases. The extremely limited overlap that we observe between liver- and fat-specific epigenetic dysregulation () suggests that each tissue responds differently to the damage and physiological insults that accumulate with age, with the potential additional contribution of distinct cellular environments during the aging process. We hypothesize that a combination of differences in mitotic activity, tissue environments and cellular susceptibilities to accumulated damage, define the reasons for cell type-specific differences in epigenomic dysregulation with age.
While this study provides us with new insights into the contribution of epigenetic dysregulation in aging, many questions remain unanswered. In prioritizing future directions, it is clear that the study in isolation of one epigenetic regulatory mechanism such as cytosine methylation is of less value than integrative studies of chromatin organization and gene expression, with orthogonal, quantitative single-locus studies to validate results at individual loci. Our results highlight the importance of testing a range of tissues and taking into account their replicative characteristics when assessing results. A focus on stem cells for analysis has the potential for greater insights than using differentiated cells, but carries the inherent problem of limited cell numbers for these genome-wide assays.
We conclude that the epigenomic dysregulation associated with aging is non-random and highly tissue-specific. Genome-wide assays focused on promoters or CG dinucleotide-dense regions would have failed to identify many of the changes we have found in this study, emphasizing the need for unbiased studies of the genome when exploring the role of cytosine methylation and the many other regulators of the epigenome in aging. More comprehensive studies of the epigenome integrated with transcriptomic assays performed on individual tissues has significant potential for identifying genes and pathways that are the targets for modification with aging, and thus insights into this aspect of the pathophysiology of aging in humans.