Age-associated Methylation Changes Research Articles

The Multi-State Epigenetic Pacemaker enables the identification of combinations of factors that influence DNA methylation.

Epigenetic clocks, DNA methylation-based predictive models of chronological age, are often utilized to study aging associated biology. Despite their widespread use, these methods do not account for other factors that also contribute to the variability of DNA methylation data. For example, many CpG sites show strong sex-specific or cell-type-specific patterns that likely impact the predictions of epigenetic age. To overcome these limitations, we developed a multidimensional extension of the Epigenetic Pacemaker, the Multi-state Epigenetic Pacemaker (MSEPM). We show that the MSEPM is capable of accurately modeling multiple methylation-associated factors simultaneously, while also providing site-specific models that describe the per site relationship between methylation and these factors. We utilized the MSEPM with a large aggregate cohort of blood methylation data to construct models of the effects of age-, sex-, and cell-type heterogeneity on DNA methylation. We found that these models capture a large faction of the variability at thousands of DNA methylation sites. Moreover, this approach allows us to identify sites that are primarily affected by aging and no other factors. An analysis of these sites reveals that those that lose methylation over time are enriched for CTCF transcription factor chip peaks, while those that gain methylation over time are associated with bivalent promoters of genes that are not expressed in blood. These observations suggest mechanisms that underlie age-associated methylation changes and suggest that age-associated increases in methylation may not have strong functional consequences on cell states. In conclusion, the MSEPM is capable of accurately modeling multiple methylation-associated factors, and the models produced can illuminate site-specific combinations of factors that affect methylation dynamics.

Local CpG density affects the trajectory and variance of age-associated DNA methylation changes

BackgroundDNA methylation is an epigenetic mark associated with the repression of gene promoters. Its pattern in the genome is disrupted with age and these changes can be used to statistically predict age with epigenetic clocks. Altered rates of aging inferred from these clocks are observed in human disease. However, the molecular mechanisms underpinning age-associated DNA methylation changes remain unknown. Local DNA sequence can program steady-state DNA methylation levels, but how it influences age-associated methylation changes is unknown.ResultsWe analyze longitudinal human DNA methylation trajectories at 345,895 CpGs from 600 individuals aged between 67 and 80 to understand the factors responsible for age-associated epigenetic changes at individual CpGs. We show that changes in methylation with age occur at 182,760 loci largely independently of variation in cell type proportions. These changes are especially apparent at 8322 low CpG density loci. Using SNP data from the same individuals, we demonstrate that methylation trajectories are affected by local sequence polymorphisms at 1487 low CpG density loci. More generally, we find that low CpG density regions are particularly prone to change and do so variably between individuals in people aged over 65. This differs from the behavior of these regions in younger individuals where they predominantly lose methylation.ConclusionsOur results, which we reproduce in two independent groups of individuals, demonstrate that local DNA sequence influences age-associated DNA methylation changes in humans in vivo. We suggest that this occurs because interactions between CpGs reinforce maintenance of methylation patterns in CpG dense regions.

Blood DNA Methylation and Aging: A Cross-Sectional Analysis and Longitudinal Validation in the InCHIANTI Study.

Changes in DNA methylation have been found to be highly correlated with aging in humans, but causes or consequences of these changes are not understood. We characterized the DNA methylomes of several hundred people in the Invecchiare in Chianti study to identify DNA sites in which percent methylation was systematically different with age. Then, we tested the hypothesis that changes of percent methylation in the same DNA sites occur longitudinally for the same DNA sites in the same subjects. We identified six differentially methylated regions in which percent methylation showed robust longitudinal changes in the same direction. We then describe functions of the genes near these differentially methylated regions and their potential relationship with aging, noting that the genes appear to regulate metabolism or cell type specificity. The nature of transcription factor binding sites in the vicinity of these differentially methylated regions suggest that these age-associated methylation changes reflect modulation of two biological mechanisms: the polycomb repressive complex 2, a protein complex that trimethylates histone H3 on lysine 27, and the transcriptional repressor CCCTC-binding factor or CTCF, both of which are regulators of chromatin architecture. These findings are consistent with the idea that changes in methylation with aging are of adaptive nature.

GRSF1 is an age-related regulator of senescence

Senescent cells that accumulate in multiple tissues with age are thought to increase pathological phenotypes. The removal of senescent cells can improve lifespan and/or healthspan in mouse models. Global hypomethylation and local hypermethylation in DNA are hallmarks of aging but it is unclear if such age-dependent methylation changes affect specific genes that regulate cellular senescence. Because mitochondria play important roles in aging and senescence, we tested if age-associated methylation changes in nuclear-encoded mitochondrial proteins were involved in regulating cellular senescence. Here, we examined the role of hypermethylation of the G-rich sequence factor 1 (GRSF1) promoter region, a mitochondrial RNA binding protein, in replication- and doxorubicin-induced cellular senescence. GRSF1 expression was lower in senescent fibroblasts, and GRSF1 knockdown induced senescence in human primary fibroblasts. These results suggest that the age-dependent hypermethylation of GRSF1 reduces its expression, which can potentially contribute to cellular senescence during aging.

Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and in vitro fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis.

Age-associated methylation changes in genomic DNA have been recently reported in spermatozoa, and these changes can contribute to decline in fertility. In a previous study, we analyzed the genome-wide DNA methylation profiles of bull spermatozoa using a human DNA methylation microarray and identified one CpG site (CpG-1) that potentially reflects age-related methylation changes. In the present study, cryopreserved semen samples from a Japanese Black bull were collected at five different ages, which were referred to as JD1-5: 14, 19, 28, 54, and 162 months, respectively, and were used for genome-wide DNA methylation analysis and in vitro fertilization (IVF). Distinct age-related changes in methylation profiles were observed, and 77 CpG sites were found to be differently methylated between young and adult samples (JD1-2 vs. JD4-5). Using combined bisulfite restriction analysis (COBRA), nine CpG sites (including CpG-1) were confirmed to exhibit significant differences in their age-dependent methylation levels. Eight CpG sites showed an age-dependent increase in their methylation levels, whereas only one site showed age-dependent hypomethylation; in particular, these changes in methylation levels occurred rapidly at a young age. COBRA revealed low methylation levels in some CpG regions in the majority of the IVF blastocyst-stage embryos derived from spermatozoa at JD2-5. Interestingly, bulls with different ages did not show differences in their methylation levels. In conclusion, our findings indicated that methylation levels at nine CpG sites in spermatozoa changed with increasing age and that some CpG regions were demethylated after fertilization. Further studies are required to determine whether age-dependent different methylation levels in bull spermatozoa can affect fertility.

Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions

BackgroundAge-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging “clock”, a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1df/df mutation, calorie restriction and rapamycin.ResultsIn wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice.ConclusionsAge-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock.

Cadmium Exposure And Age-Associated Dna Methylation Changes In Non-Smoking Women From Northern Thailand

Introduction: Cadmium (Cd) exposure initiates cellular processes that promote aging and disrupts methylation globally. Aging is associated with DNA methylation changes, and age-associated methylation markers may serve as a biomarker of biologic age. While Cd exposure increases risk of age-related chronic diseases, we do not know if Cd mediates this effect via age-associated methylation changes. We analyzed age-associated methylation and assessed the difference between age predicted by methylation and age (Δage) in non-smoking northern Thai women exposed to Cd via environmental contamination of food. Methods: We analyzed methylation in =450,000 CpG sites in 40 non-smoking women age 40-80. We classified these women as high (HE) and low (LE) Cd exposed using a cutoff of 3 ug/L for urine Cd and matched by age within five years. We predicted methylation age (mAge) using two published methods by Horvath and Hannum et al. We analyzed differences by Cd using limma, paired t-tests, and linear mixed models. Results: Mean urine Cd was 1.0 and 16.3 ug/L in LE and HE. We identified eight age-associated differentially methylated markers in ELOV2, KLF14, PCDHB1, FHL2, FAM84B, DUSP22, and IL21R (adjusted p

Acceleration of age-associated methylation patterns in HIV-1-infected adults.

Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x10-200 and 0.47, p<1x10-200. Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage= 0.007088, p=2.08 x 10-9; βHIV= 0.099574, p=0.0011; Data set 2: βage= 0.008762, p=1.27x 10-5; βHIV= 0.128649, p= 0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10-6, odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular and molecular mechanisms. These results are an important first step for finding potential therapeutic targets and novel clinical approaches to mitigate the detrimental effects of both HIV-1-infection and aging.

Age-related DNA methylation changes for forensic age-prediction.

There is no available method of age-prediction for biological samples. The accumulating evidences indicate that DNA methylation patterns change with age. Aging resembles a developmentally regulated process that is tightly controlled by specific epigenetic modifications and age-associated methylation changes exist in human genome. In this study, three age-related methylation fragments were isolated and identified in blood of 40 donors. Age-related methylation changes with each fragment was validated and replicated in a general population sample of 65 donors over a wide age range (11-72years). Methylation of these fragments is linearly correlated with age over a range of six decades (r = 0.80-0.88). Using average methylation of CpG sites of three fragments, a regression model that explained 95% of the variance in age was built and is able to predict an individual's age with great accuracy (R (2 )= 0.93). The predicted value is highly correlated with the observed age in the sample (r = 0.96) and has great accuracy of average 4years difference between predicted age and true age. This study implicates that DNA methylation can be an available biological marker of age-prediction. Further measurement of relevant markers in the genome could be a tool in routine screening to predict age of forensic biological samples.

Aging of blood can be tracked by DNA methylation changes at just three CpG sites

BackgroundHuman aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age.ResultsWe perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs - located in the genes ITGA2B, ASPA and PDE4C - were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita - two diseases associated with progressive bone marrow failure and severe telomere attrition - are predicted to be prematurely aged.ConclusionsOur epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.

Specific age-associated DNA methylation changes in human dermal fibroblasts.

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner.