textCytosine methylation of CpG dinucleotides is an important epigenetic mark that regulates gene expression in humans. While methylation patterns in extant populations have been widely studied, few studies have attempted to analyze methylation in ancient DNA. Indeed, it was only recently shown that methyl groups can be preserved in ancient DNA. However, it is unknown how often methylation patterns can be recovered from ancient samples with preserved nuclear DNA. If they are frequently preserved, it may ultimately be possible to infer patterns of gene activity at the population level in ancient times. In this study, I assessed the preservation of cytosine methylation in ancient DNA from the remains of 30 prehistoric Native Americans from California, Illinois, Kentucky, and Mexico. These samples were previously shown to contain endogenous mitochondrial and nuclear DNA. I analyzed the cytosine methylation states of CpG-rich retrotransposons, which are epigenetically inactivated by cytosine methylation in humans. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite. Bisulfite products were pyrosequenced, and C-to-T conversions at potentially methylated CpG dinucleotides were quantified. I found that cytosine methylation is readily recoverable from human remains with preserved nuclear DNA from various localities over the time depth tested (~6000 years). This study presents the first direct evidence of cytosine methylation in ancient human remains, and suggests that it may be possible to analyze patterns of gene activity in ancient populations.Anthropolog
