Distinct DNA methylomes of newborns and centenarians

Abstract

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine--phosphate--guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

Fig. 1.

WGBS of a newborn (NB) and a centenarian (Y103) individual. (A) Circos representation of genome-wide DNA methylation levels in the NB, Y26, and Y103 individuals. Average levels for all of the CGs in 297 10-Mbp-wide windows. Inner track indicates the magnitude of the difference between the Y103 and the NB individual for each window (color scale and red line). Average methylation levels in all of the regions are expressed as β-values (0–1) and are colored blue. (B) Total number of methylated CpG sites and the CpG methylation level (%) in the DNA from the newborn (NB), an intermediate 26-y-old sample (Y26), and the centenarian sample (Y103). (C) Illustrative CpG methylation levels for Y103 (red line) and NB (blue line) in chromosomes 2 and 8. (D) The curves of the distance correlation between the methylation status of neighboring CpG sites of NB (blue line), Y26 (green line), and Y103 (red line) samples determined by WGBS. A more pronounced declining curve indicates lower correlation in terms of the methylation status of nearby CpGs. (E) Mean CpG methylation levels among different genomic sequences in the centenarian and the newborn individuals. (F) Mean CpG methylation levels among promoters in the centenarian and the newborn individuals according to the presence or absence of a CpG island. **P < 0.01 in the Fisher’s exact t test. The mean methylation level is calculated by the number of total methylated reads divided by the numbers of total reads covering CpG sites located in the sum of the feature analyzed.

Fig. 2.

DMRs of NB and Y103. (A) Circos representation of average methylation levels for all of the CGs in the DMRs in chromosome 2, indicating whether the region was methylated (red) or unmethylated (green) in the Y103 sample relative to the NB sample. Regions are equally spaced around the figure, but their original locations in the genome are indicated by gray lines. (B) DMR distribution among different genomic sequences. (C) Distribution of DMRs according to the direction of the DNA methylation change, the type of promoter (with or without a CpG island), and the reported expression pattern.

Fig. 3.

WGBS data validation with a 450-K CpG site DNA methylation microarray. (A) Comparison of CpG methylation differences between the NB and Y103 obtained from WGBS (x-axis) and 450-K array (y-axis) technology. Displayed are all differentially methylated CpG sites derived from the WGBS approach that were also present on the 450-K array (3,205). The threshold of 0.20 change in β-values is indicated (broken line). (B) The curves of the distance correlation between the methylation status of the neighboring CpG sites of NB (blue line) and Y103 (red line) samples determined by the 450-K microarray. A more pronounced declining curve indicates lower correlation in terms of the methylation status of nearby CpGs.

Fig. 4.

Extension of the WGBS data to a comprehensive set of newborns and nonagenarians and the observed overlap in premature aging disorders. (A) The curves of the distance correlation between the methylation status of the neighboring CpG sites of newborns (blue line) and nonagenarians (red line) samples determined by the 450 K microarray. A more pronounced declining curve indicates lower correlation in terms of the methylation status of nearby CpGs. (B) Unsupervised hierarchical clustering of the 283,579 CpGs present in the 450 K methylation array (after exclusion of SNP-associated and X and Y chromosome CpG sites) in the newborn and nonagenarian groups. (C) Schematic overview of the assessment of differentially methylated CpG sites between the initial newborn and 103-y-old sample and the cohort of 19 newborns and 19 nonagenarians. (D) Hierarchical clustering approach using the 1,149 WGBS-derived dmCpGs in the newborn and nonagenarian groups. (E) Supervised clustering analysis with the 165 DMRs (214 dmCpG dinucleotides) that distinguished the two groups. (F) Hierarchical clustering approach using the additional 5,774 450K-dmCpG sites in the newborn and nonagenarian groups. (G) (Upper) The 450K-dmCpG distribution among different genomic sequences; (Lower) Distribution of the 450K-dmCpGs according to the direction of the DNA methylation change and the type of promoter (with or without a CpG island).