Aging and Caloric Restriction Modulate the DNA Methylation Profile of the Ribosomal RNA Locus in Human and Rat Liver

Abstract

A growing amount of evidence suggests that the downregulation of protein synthesis is an adaptive response during physiological aging, which positively contributes to longevity and can be modulated by nutritional interventions like caloric restriction (CR). The expression of ribosomal RNA (rRNA) is one of the main determinants of translational rate, and epigenetic modifications finely contribute to its regulation. Previous reports suggest that hypermethylation of ribosomal DNA (rDNA) locus occurs with aging, although with some species- and tissue- specificity. In the present study, we experimentally measured DNA methylation of three regions (the promoter, the 5' of the 18S and the 5' of 28S sequences) in the rDNA locus in liver tissues from rats at two, four, 10, and 18 months. We confirm previous findings, showing age-related hypermethylation, and describe, for the first time, that this gain in methylation also occurs in human hepatocytes. Furthermore, we show that age-related hypermethylation is enhanced in livers of rat upon CR at two and 10 months, and that at two months a trend towards the reduction of rRNA expression occurs. Collectively, our results suggest that CR modulates age-related regulation of methylation at the rDNA locus, thus providing an epigenetic readout of the pro-longevity effects of CR.

Keywords: DNA methylation; aging; caloric restriction; liver; ribosomal RNA.

Figure 1

The rDNA locus undergoes hypermethylation during aging in rat liver. (A) Lineplots of DNA methylation profiles of the RiboPromoter, 18S and 28S amplicons in liver tissue from rats fed ad libitum for two, 10, and 18 months. For each CpG unit, mean methylation and standard deviation across four biological replicates for each time point are reported. (B) Coefficients of Pearson’s correlation between DNA methylation and age for each CpG site in the three target regions. (C) Scatterplots of DNA methylation values vs. age for the CpG unit most associated with age in each amplicon. *: p-value < 0.05; **: p-value < 0.01.

Figure 2

The rDNA locus undergoes hypermethylation during aging in human liver. (A) Coefficients of Pearson’s correlation between DNA methylation and age for each CpG site in the 3 target regions. (B) Scatterplots of DNA methylation values vs. age for the CpG unit most associated with age in each amplicon. *: p-value < 0.05; **: p-value < 0.01.

Figure 3

The rDNA locus undergoes hypermethylation in livers from caloric restriction (CR) fed rats. Lineplots of DNA methylation profiles of the RiboPromoter, 18S and 28S amplicons in liver tissue from rats fed ad libidum (AL) and under CR for two, 10, and 18 months. For each CpG unit, mean methylation and standard deviation across four biological replicates for each time point are reported.

Figure 4

45S rRNA precursor and Rrn3 expression do not significantly change with aging in rat livers. (A) Scatterplot of 45S expression vs. age in rat livers. (B) Scatterplot of 45S expression vs. Rrn3 expression in rat livers. Samples are colored according to age, using the colors used in panel (A). (C) Scatterplot of Rrn3 expression vs. age in rat livers.

Figure 5

Effect of CR on the expression of 45S and of Rrn3 in aging rat livers. For each time point (two, 10, and 18 months) the expression of 45S and Rrn3 transcripts in AL and CR rat livers is reported as boxplots. Values from individual animals are reported as dots.