A Combined Genetic and Epigenetic Approach to Understanding Human Neocortical, Behavioral, and Medical Circadian Rhythms

Background: Circadian rhythms – intrinsic 24-hour biological rhythms - have a major impact on human health and behavior, and play an important role in neurological diseases such as dementia and epilepsy. In model organisms, circadian rhythms are generated in neurons of the suprachiasmatic nucleus (SCN) by a transcription-translation feedback cycle involving a set of evolutionarily conserved “clock” genes. Similar clocks are present in other tissues too. These tissue-specific clocks are entrained by the SCN and are responsible for circadian modulation of tissue physiology. They do so in part through tissue-specific 24-hour cycles of epigenomic modification, driving 24-hour cycles of gene expression. There is little data about circadian genomic, epigenomic, and transcriptomic mechanisms in the human neocortex – an important gap because the neocortex is central to neurological diseases like dementia and epilepsy, and understanding the mechanisms linking the neocortical clock to brain physiology is key to understanding the impact of circadian rhythms on these diseases and their treatment. Moreover, although many circadian traits appear to be heritable, genetic association studies to date have identified few gene variants robustly associated with objectively quantified human circadian behavior, or with changes in circadian rhythmicity of the conserved molecular clock.

Methods: We undertook a series of studies to examine objectively quantified circadian rhythms of human behavior, circadian rhythms of clock gene DNA methylation and RNA expression human neocortex, and genetic variants influencing these rhythms. 1) We quantified DNA methylation at 112 CpG sites in or near 6 canonical clock genes - PER2, PER3, CRY1, CRY2, and ARNTL - using Illumina Infinium HumanMethylation450 microarray data from neocortical samples from 490 deceased individuals in 2 cohort studies of aging (the Religious Orders Study and the Rush Memory and Aging Project) and parameterized methylation level at each CpG site as a function of time of death using cosine curves. 2) We quantified transcript abundance for these genes using Illumina Human HT-12 Expression microarray data from a subset of 490 of these individuals and parameterized transcript level as a function of time of death using cosine curves. 3) We performed a candidate-gene association study with replication, evaluating associations between polymorphisms in homologs of evolutionarily conserved clock genes and the timing of behavioral rhythms measured by actigraphy. For validated polymorphisms, we evaluated associations with rhythms of transcript expression, and time of death in additional cohorts.

Results: Significant daily rhythms of methylation were seen in 52/112 CpG sites (p<0.05), and in the expression of PER2 (p=3.1x10-4), PER3 (p=2.5x10-4), CRY1 (p=7.0x10-4), ARNTL (p=5.9x10-5), and CLOCK (p=7.3x10-3) with the timings of peak transcript abundance mirroring those seen in other diurnal mammals. The timing of the nadir of methylation was site specific. For rhythmic CpG sites within the gene body, the timing of the nadir of methylation clustered between 16:00 and 22:00, irrespective of the timing of transcript abundance. However, for sites upstream of the promoter region and in the 5’UTR, the timing of the nadir of methylation was roughly in-phase with the timing of peak abundance of the corresponding transcript showing temporal correlation between hypomethylation and expression. Meanwhile, in a parallel genetic association study, rs7221412, a common polymorphism near period homolog 1 (PER1), was associated with the timing of activity rhythms in discovery and replication cohorts (joint p=2·1x10-7). Mean activity timing was delayed by 67 minutes in rs7221412GG vs. rs7221412AA homozygotes. rs7221412 also showed a suggestive relationship with the circadian phase of the expression of multiple clock genes in human neocortex, and an interesting association with time of death (p=0.015) in which rs7221412GG individuals had a mean time of death nearly seven hours later than rs7221412AA/AG.

Conclusions: Daily rhythms of clock gene expression and parallel rhythms of DNA methylation at nearby sites are present in human cerebral cortex and have characteristic site-specific phase relationships to each other and to global circadian rhythms. Moreover, a common polymorphism near PER1 is associated with the timing of human behavioral rhythms, and shows evidence of association with the timing of rhythms of clock gene expression, and with time of death. Cycles of DNA methylation and gene transcription may play a role in the regulation of the circadian clock in human neocortex. Common gene variants associated with the timing of human behavior and human medical outcomes may act in part by influencing these neocortical rhythms. A multi-level “-omic” approach to neocortical and behavioral circadian rhythms may identify novel genetic regulators of the human neocortical clock and delineate potential mechanisms linking the clock to neocortical physiology. In so doing, it may lay the foundation for clinical efforts to leverage circadian biology to promote neurological health.