Ribosomal DNA harbors an evolutionarily conserved clock of biological aging

Abstract

The ribosomal DNA (rDNA) is the most evolutionarily conserved segment of the genome and gives origin to the nucleolus, an energy intensive nuclear organelle and major hub influencing myriad molecular processes from cellular metabolism to epigenetic states of the genome. The rDNA/nucleolus has been directly and mechanistically implicated in aging and longevity in organisms as diverse as yeasts, Drosophila, and humans. The rDNA is also a significant target of DNA methylation that silences supernumerary rDNA units and regulates nucleolar activity. Here, we introduce an age clock built exclusively with CpG methylation within the rDNA. The ribosomal clock is sufficient to accurately estimate individual age within species, is responsive to genetic and environmental interventions that modulate life-span, and operates across species as distant as humans, mice, and dogs. Further analyses revealed a significant excess of age-associated hypermethylation in the rDNA relative to other segments of the genome, and which forms the basis of the rDNA clock. Our observations identified an evolutionarily conserved marker of aging that is easily ascertained, grounded on nucleolar biology, and could serve as a universal marker to gauge individual age and response to interventions in humans as well as laboratory and wild organisms across a wide diversity of species.

Figure 1.

Ribosomal DNA methylation is strongly associated with age. (A) Age-associated hypermethylation of rDNA CpGs in mice. Spearman's correlation coefficients with age (ρ_age) for CpGs along the rDNA sequence. Red dots indicate CpGs with significant positive correlation with age (ρ_age > 0, FDR < 0.01); pink and light blue dots denote nonsignificant CpGs (FDR > 0.01) with positive and negative coefficients, respectively. The green circle indicates CpG 7044. (B) Scatterplot shows the correlation between CpG 7044 methylation and age (ρ = 0.78, P < 2.2 × 10⁻¹⁶). (C) The distribution of ρ_age for CpGs within the rDNA and across the whole genome (Wilcoxon rank-sum test, P < 2.2 × 10⁻¹⁶). (D,E) Cumulative distribution of correlation coefficients for various genomic elements. CpGs within CpG islands (CGIs) and bivalent chromatin have significantly higher ρ_age than the genome-wide background, although both of them are significantly lower than rDNA CpGs (P < 2.2 × 10⁻¹⁶). Repetitive elements, including L1, L2, Alu (B elements), and mammalian-wide interspersed repeats (MIR), tend to be hypomethylated during aging (P < 2.2 × 10⁻¹⁶) relative to the genome-wide background. All features on D, except for intron, show a shift to the right of the genome-wide background. All features on E, except for the rDNA, show a shift to the left of the genome-wide background. Also see Supplemental Figure S5.

Figure 2.

Building the rDNAm age clock. (A,B) Example of two rDNA methylation clock models: (A) Model 1; (B) Model 2. Note that the training and testing subsets are reversed in the two models. (C,D) Performance of 20,000 models trained and tested on randomly split subsets of the mice data set. (C) Correlation coefficients (ρ) between the predicted age (i.e., rDNAm age) and chronological age of the test subsets were plotted against the number of clock CpGs of each model. (D) The median absolute errors (MAEs) of the rDNAm ages were plotted against the number of clock CpGs of each model. (E) Location and weights of the 72 clock sites identified by the best-fitted model. The three gray blocks represent the 18S, 5.8S, and 28S components (from left to right). The color coding represents the strength of age association in each site.

Figure 3.

rDNA methylation and the rDNAm clock are responsive to interventions that modulate life-span. (A) C57BL/6 mice subjected to calorie restriction (CR) (starting at 14 wk old) displayed lower rDNAm age than mice fed ad libitum (AL) (one-tailed t-test of the differences between rDNAm age and chronological age, P = 1.17 × 10⁻⁹). CR mice were at four age stages (10, 18, 23, and 27 mo old, each with five samples). The theoretical line is shown in dashed gray. The black and blue lines show the regression line for the relationship between chronological and biological age of control and CR mice, respectively. (B) Derived iPSC cell lines have significantly lower rDNAm ages than their progenitor kidney and lung fibroblasts: (*) P ≤ 0.039; three samples in each group. (C–E) Correlations between the ρ_age of each of the 816 rDNA CpGs used to train the rDNAm clock and their change in methylation caused by interventions. (C) C57BL/6 strain 27-mo-old CR mice were considered (versus ad libitum 26-mo-old mice, i.e., the ones with the closest ages). (D,E) Derived iPSC cell lines relative to fibroblast progenitors. All samples are from the Petkovich set (Petkovich et al. 2017).

Figure 4.

The ribosomal rDNAm clock is conserved between distantly related taxa. (A) Phylogenetic tree of seven vertebrate species with the number of homologous CpGs in each species (relative to human). (B) Interspecific mouse-canid clock models built using homologous CpGs in the rDNA yielded significantly better performance than those built using homologous genome-wide CpGs. Here, the model is trained in one species and applied to the other species. The fit is measured as the correlation coefficient (ρ) between rDNAm age and chronological age in the testing species. (C) Homologous CpGs from the rDNA show conserved age association (ρ_age) between mice and canids (ρ = 0.49, P = 1.88 × 10⁻⁶). (D) Homologous CpGs elsewhere in the genome show age association that is weakly correlated between mice and canid (ρ = 0.09, P = 7.01 × 10⁻¹⁶).