Musica Universalis is an ancient philosophical concept that claims the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that men make were mere an approximation of this larger harmony of the universe. Besides music, electromagnetic waves such as light and electronic signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until very recently that the same harmonic resonance was discovered to also exist in biological systems. Intriguingly, contrary to the traditional belief that biological system is either at constant homeostasis (not rhythmic) or cycling with a single frequency, it is now appreciated that most biological systems have no homeostatic “set point”, but rather oscillate as composite rhythms consisting of a series of superimposed oscillations. These oscillations often cycle at different harmonics of the circadian rhythm (~24h), and among them the ~12h oscillations are found very prevalent. I posit biological rhythms are also “Musica Universalis”: while the circadian rhythm is synchronized to the 24h light/dark cycle coinciding with the Earth’s rotation, the mammalian 12h clock may have evolved from the circatidal clock, which is in turn entrained by the 12h tidal cues orchestrated mainly by the moon.
In collaboration with Dr. Antoulas, the eigenvalue/pencil method was developed to uncover all superimposed oscillations from a time series dataset. Unlike most of the current cycling transcripts-identification methodologies that require the user to define a narrow period range, the eigenvalue/pencil method does not pre-assign a period and thus permits the identification of all superimposed oscillations in an unbiased manner. For example, deconvolution of mouse hepatic Pck1 gene mRNA expression by the eigenvalue/pencil method revealed superimposed harmonic oscillations cycling at ~24h, 12h, 8h and 4h.
When applying the eigenvalue/pencil method to all transcripts and metabolites in mouse liver, oscillations cycling at different harmonics of circadian rhythm (8h, 12h and 24h) were identified. Gene Ontology analysis of 12h-cycling mouse transcripts revealed enrichment in the entire genetic information flow process including mRNA metabolism and protein processing and unfolded protein response pathways, among many others (A). In addition to liver, 12h-cycling transcripts were also found predominantly in brown adipose tissue (BAT), skeletal muscle, white adipose tissue (WAT), aorta, lung, adrenal gland and heart, indicating a very prevalent presence of 12h-clock (B).
Besides mice, 12h rhythms of proteostasis and mRNA metabolism gene expression are also observed in primates, including hippocampus and cerebellum regions of baboons and peripheral white blood cells and dorsolateral prefrontal cortex of humans.
Consistent with the strong enrichment of ER stress and unfolded protein response (URP) pathways in 12h-cycling hepatic transcriptome, 12h cycling of UPR transcription factor XBP1s and ATF4 was found in mouse liver. Furthermore, 12h-cycling of XBP1s expression was found in tunicamycin-synchronized MEFs and is independent from the circadian clock. Both the total level of Xbp1 mRNA (the combined level of Xbp1s and Xbp1us) and its splicing efficiency exhibited robust 12h rhythms both in mouse liver in vivo and in MEFs in vitro. Knock-down of Xbp1 further abolished cell-autonomous 12h rhythms of Eif2ak3 (a 12h-clock target gene) promoter-driven dGFP intensity in single MEF. In XBP1 liver-specific knockout mice, global ~12h, but not circadian rhythms of gene expression was either abolished or dampened.
We recently showed that liver-specific ablation of the 12h-oscillator due to XBP1 liver ablation is associated with substantially accelerated liver aging, characterized by a much earlier onset of fatty liver, mitochondria dysfunction, increased ER stress and dysregulated lipid metabolism observed in 5-8 months old XBP1 LKO mice. Since the ablation of the 12h transcriptome precedes the liver aging phenotype, we speculate that the 12h-oscillator dysregulation could drive liver aging. Since the hepatic core circadian clock remains intact both during aging and in XBP1 LKO mice, 12h-oscillator dysregulation likely can cause hepatic aging independently from the circadian clock.
We postulate that the mammalian 12h clock evolved from the ancient circatidal clock of marine animals, which adapt their behaviors to the 12h rhythms of ebb and flow of the tides. Conserved 12h rhythms of gene expression are found in marine animals possessing dominant circatidal clocks (such as a limpet Cellana rota), nematode, zebrafish, fly, mouse and baboon.
With the vast amount of high-resolution temporal transcriptome and cistrome data available (high resolution time series data), it is possible to infer the gene regulatory network of mammalian 12h-clock computationally. Combining eigenvalue/pencil-based identification of robust 12h oscillating transcriptome, motif analysis of gene regulatory region of 12h-osillating genes and gene regulatory network inference by decision tree-based regression models, we can have a first glimpse of the 12h-clock gene regulatory network.
We are currently continuing to refine our network inference in multiple mouse tissues, with the ultimate goal of constructing a network that is most common to most tissues. Computationally predicted network will be experimentally validated by systemic transcriptome/cistrome profiling in mouse tissues in vivo and time lapse imaging in vitro in single cell.
Ultimately, we aim to mathematically model the 12h-oscillator with ODEs so that we can have a theoretical framework of the 12h oscillator.
After the computational inference of potential TFs and epigenetic regulators of the mammalian 12h-clock, we will then experimentally validate their implication in 12h-clock control by performing hepatic temporal ChIP-Seq and RNA-Seq analysis in wild-type mice and mice lacking hepatic expression of these factors. Systemic profiling will be complemented with detailed biochemical and molecular biology assays to elucidate the detailed molecular mechanism of how these TFs/co-factors transcriptionally regulate the 12h-clock.
We aim to elucidate the function and prevalence of 12h oscillator in additional tissues in mice, with a particularly focus on the potential roles of 12h oscillator in the central nervous system.
We are interested in investigating the mechanisms of how the 12h oscillator senses and responds to metabolic stress and how the genetic information flow alters as a result.