If asked what are the differences between a grandfather and his newborn granddaughter, I would reply with the obvious ones: size (the grandfather is larger than his granddaughter), condition of the skin (babies have soft, smooth skin and elders have age spots and wrinkles) and life expectancy. Other visual cues may seem more similar than different. For example, grandfather and granddaughter may both lack hair on their heads or need assistance to move from one place to another. However, both baby and elder are a product of the genes expressed from their genome even if the exact sequence is not identical between them. Because genes are expressed differently over a human’s lifetime, Heyn et al. decided to examine the methylation profile in the genomes of newborns and individuals 89 years old or older.
The research group decided to first study the genomes of two males, a newborn and a 103-year-old man. This age extreme was expected to show the greatest methylation difference if there was any that existed between the two genomes. Specifically, they wanted to look at the methylation state of cytosine in CpG sites, locations that influence how active a gene is. Promoter regions tend to be CpG rich and the greater the methylation at the promoter site, the lower the expression from the downstream gene. Genomic DNA was isolated from CD4+ T cells from newborn cord blood or adult whole blood and the methylation state studied using whole-genome bisulfite sequencing (WGBS) and a 450,000 CpG DNA methylation microarray. Bisulfite reacts with cytosine but not methyl cytosine. The resulting DNA can be sequenced to identify methylated cytosine, while the microarray has numerous individual CpG regions attached to the surface of a slide to detect changes in methylation of these sites throughout a genome.
The WGBS uncovered that the 103-year-old man’s DNA had 494,595 fewer methylated CpG dinucleotides (mCpGs) than did DNA from the newborn. Furthermore, CpG methylation in the elderly man was lower overall (73%) compared to the newborn’s DNA (80.5%). The researchers also compared the methylation state of a 26 year old’s genome and found 77.8% CpG methylation, a level between the newborn and the centenarian. Upon closer examination of promoters that are CpG rich and CpG poor, Heyn et al. discovered CpG-poor promoters were more likely to be unmethylated and CpG-rich promoters were more methylated in the 103-year-old man compared to the newborn. Further analysis of identical CpG sites between the newborn and centenarian found 17,930 differentially methylated regions (DMRs) distributed across all chromosomes. Comparing the DMRs also showed 87% of the regions had lost methylation versus 13% that gained methylation in the elderly man compared to the newborn. The hypermethylated DMRs corresponded to CpG islands, rich in CpG sites, while researchers observed 83% of CpG-poor promoters and 55% of CpG-island promoters were hypomethylated. This reduced methylation increased gene expression in eight tissue-specific genes assessed.
When the newborn and centenarian samples were assessed with the 450,000 CpG DNA methylation microarray, the results correlated to those seen with WGBS, and the double-validated DMR (found in both WGBS and the CpG microarray) were confirmed for five randomly selected promoters. The study was then expanded to 19 newborns and 19 elderly adults (89–100 years of age) and the methylation status assessed with the 450,000 CpG DNA methylation microarray, excluding CpG sites that were sex-chromosome specific. Analysis of the data generated from these sample groups found 214 CpG DMRs that could distinguish the two groups. Again, these differentially methylated sites were confirmed with bisulfite conversion and sequencing, and gene expression analysis. This larger sample size also confirmed that the changes most often seen in the elderly samples were decreased methylation compared to the newborn samples, and CpG-poor promoters had 93% of CpG sites that were unmethylated while CpG-island promoters had 58% of CpG sites methylated. Analysis of samples from adults averaging 59.8 years of age found 70% of the CpG sites that distinguished newborn from elderly had intermediate levels of methylation.
This study published in the Proceedings of the National Academy of Sciences USA was the first to compare the methylation status of genomes from newborns and individuals 89 years of age or older. The age disparity allowed the researchers to examine the differences in methylation and discover that the decrease in methylation seen in elderly compared with newborns are accumulated over the lifetime and can increase expression of genes that have roles in cancer and other conditions found more commonly in older adults than babies. The DMRs found by these researchers add to the epigenetic data related to aging and offer new possibilities in exploring the role of methylation in aging and age-related diseases.
Heyn, H., Li, N., Ferreira, H.J., Moran, S., Pisano, D.G., Gomez, A., Diez, J., Sanchez-Mut, J.V., Setien, F., Carmona, F.J., Puca, A.A., Sayols, S., Pujana, M.A., Serra-Musach, J., Iglesias-Platas, I., Formiga, F., Fernandez, A.F., Fraga, M.F., Heath, S.C., Valencia, A., Gut, I.G., Wang, J.and Esteller, M. (2012). Distinct DNA methylomes of newborns and centenarians. Proceedings of the National Academy of Sciences of the United States of America PMID: 22689993