24-Hour Rhythms of DNA Methylation and Their Relation with Rhythms of RNA Expression in the Human Dorsolateral Prefrontal Cortex

Abstract

Circadian rhythms modulate the biology of many human tissues, including brain tissues, and are driven by a near 24-hour transcriptional feedback loop. These rhythms are paralleled by 24-hour rhythms of large portions of the transcriptome. The role of dynamic DNA methylation in influencing these rhythms is uncertain. While recent work in Neurospora suggests that dynamic site-specific circadian rhythms of DNA methylation may play a role in modulating the fungal molecular clock, such rhythms and their relationship to RNA expression have not, to our knowledge, been elucidated in mammalian tissues, including human brain tissues. We hypothesized that 24-hour rhythms of DNA methylation exist in the human brain, and play a role in driving 24-hour rhythms of RNA expression. We analyzed DNA methylation levels in post-mortem human dorsolateral prefrontal cortex samples from 738 subjects. We assessed for 24-hour rhythmicity of 420,132 DNA methylation sites throughout the genome by considering methylation levels as a function of clock time of death and parameterizing these data using cosine functions. We determined global statistical significance by permutation. We then related rhythms of DNA methylation with rhythms of RNA expression determined by RNA sequencing. We found evidence of significant 24-hour rhythmicity of DNA methylation. Regions near transcription start sites were enriched for high-amplitude rhythmic DNA methylation sites, which were in turn time locked to 24-hour rhythms of RNA expression of nearby genes, with the nadir of methylation preceding peak transcript expression by 1–3 hours. Weak ante-mortem rest-activity rhythms were associated with lower amplitude DNA methylation rhythms as were older age and the presence of Alzheimer's disease. These findings support the hypothesis that 24-hour rhythms of DNA methylation, particularly near transcription start sites, may play a role in driving 24-hour rhythms of gene expression in the human dorsolateral prefrontal cortex, and may be affected by age and Alzheimer's disease.

Author Summary

Circadian rhythms are intrinsic 24-hour biological rhythms that influence many aspects of human biology, including normal and abnormal human brain functions such as cognition and seizures. Circadian rhythms are maintained by a near 24-hour feedback loop mediated by a series of “clock” genes that are similar across species, including humans. However, the specific mechanisms underlying the circadian regulation of gene transcription are unknown. DNA methylation is an epigenetic mechanism that can influence gene expression without changes in DNA sequence. The 24-hour rhythms of DNA methylation are one mechanism contributing to 24-hour rhythms of clock gene expression in fungi. However, this has not been demonstrated in mammals including humans. In this study, we examined levels of DNA methylation at>400,000 sites across the genome in the brains of 738 human subjects and showed significant 24-hour rhythms of DNA methylation. Moreover, we showed that for specific locations of DNA methylation site, these rhythms of methylation were linked to rhythms of gene expression. This is important because it suggests that circadian rhythms of DNA methylation may be an important mechanism underlying circadian rhythms of gene expression in the human brain, and hence circadian rhythms of normal and abnormal brain function.