Genetic basis of circadian rhythms and sleep disorders

Project Abstract Our long-term goal is to understand the genetic and molecular organization of circadian rhythms in mammalian models and humans, and the molecular mechanisms underpinning disorders of circadian rhythms and sleep. In doing so, we will provide important insights for treating a wide variety of diseases in which sleep disorders are major causes, symptoms, or comorbidities. We are pursuing three research areas under this common goal. 1) The genetic basis of human chronotypes and sleep disorders: Despite a clear causal link between genetic polymorphism in clock genes and sleep disorders, systematic investigation of pathogenic mutations in human clock genes has not been conducted for two reasons: The first is the abundance of natural genetic polymorphisms. There are thousands of known polymorphisms in major clock genes, a subset of which may affect chronotypes and sleep disorders. The second is the historical reliance on rodent models for functional assessment, which are time- and resource-intensive. To address this gap, we developed a cell-based in vivo- like platform that allows us to screen and characterize dozens of hypothesis-driven mutations along with natural human mutations in a cost- and labor-effective manner. Using our system, we identified novel mechanisms and mutations that can cause dramatically altered rhythms and sleep cycles. During the course of analyzing dozens of mutations in pacemaker clock genes, we found common themes that allow us to identify potential pathogenic mutations and predict their phenotypes. 2) The molecular link between the circadian clock and sleep homeostasis: Sleep is regulated by two distinct but interconnected mechanisms: sleep homeostasis and the circadian clock. Optimal sleep quality requires alignment of these two sleep-regulating pathways, and disruptions in either one can lead to sleep disorders. However, the mechanistic links between the circadian clock and sleep homeostasis have been largely unexplored. Recently, we serendipitously found that a Per2-driven clock in Per1 knockout (KO) mice behaves very differently from a wild-type (wt) clock and from a Per1-driven clock in Per2 KO mice when they are perturbed by long light pulses. Per1 KO mice showed dramatic phase shifting, called type 0 resetting, while wt and Per2 KO mice exhibited weak (type 1) resetting. Per1 KO mice always reset to CT12, the time of activity onset or lowest sleep pressure, regardless of when the light pulses were given. The Per2-driven oscillator seems to measure or regulate temporal sleep pressure, in addition to driving circadian rhythms. We propose to continue this line of investigation to unravel the molecular link between the clock and sleep homeostasis. 3) Identification of novel clock genes: We engineered a human cell line with a functioning circadian clock (U2OS) to express fluorescent protein-fused clock proteins from endogenous loci and thus report expression of endogenous clock genes. With powerful CRISPR lentiviral libraries targeting all human genes, we will identify novel candidate genes that affect expression of the reporter clock genes by FACS and high-throughput sequencing.