Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging [Bisulfite-Seq]

Circadian rhythmicity governs a remarkable array of fundamental biological functions and is mediated by cyclical transcriptomic and proteomic activities. Epigenetic factors are also involved in this circadian machinery; however, despite extensive efforts, detection and characterization of circadian cytosine modifications at the nucleotide level have remained elusive. In this study, we report that a large proportion of epigenetically variable cytosines show a circadian pattern in their modification status in mice. Importantly, the cytosines with circadian epigenetic oscillations significantly overlapped with the cytosines exhibiting age-related changes in their modification status. Our findings suggest that evolutionary advantageous processes like circadian rhythmicity can also contribute to an organism's deterioration. Overall design: The animals were singly housed with ad libitum access to food, water, and a 17 cm diameter running wheel mounted in the cage. The animals were entrained to a 24-hr light-dark cycle maintained at 12 hr light and 12 hr dark (LD 12:12), where Zeitgeber time 0 (ZT0) refers to light onset. Mice were euthanized for tissue collection at 9, 15, or 25 months of age by cervical dislocation. Harvested tissues were snap frozen in liquid nitrogen and stored in -80 °C prior to downstream processing. The sequencing experiment was performed in technical triplicates using padlock bisulfite/oxybisulfite sequencing (22306810).