Project Summary/Abstract
Genetic disruptions such as pathogenic single-nucleotide polymorphisms (SNPs) in clock genes can perturb
circadian rhythms and cause sleep disorders. For example, the tau mutation in the CK1ε gene causes dramatic
shortening of the wake-sleep cycle in animals, ~20 hrs instead of normal 24 hrs. The same mutation has been
reported as a SNP in humans. Because the circadian clock mechanism is conserved across mammalian
species, affected humans would be predicted to have the same altered sleep cycle. Structural and biochemical
assays have predicted many potentially pathogenic mutations exist for clock genes. However, we do not yet
understand the in vivo significance of these potentially pathogenic mutations. A critical bottleneck in studying
the pathogenesis of genetic disruptions is lack of an efficient in vivo system that uses cell models instead of
resource-intensive, live animal models.
Aim 1. Develop an efficient platform to study mammalian clock mechanisms and identify pathological
mutations. To test the hypothesis that functionality of SNPs in clock genes can be studied in a human cell-
based platform, we generated endogenous Per1-luc and Per2-luc reporters in U2OS cells where diverse SNPs
will be generated and assessed accurately. We have validated the system by confirming consistent knockout
phenotypes between our reporter cells and mice, and reproducing similar phenotypes with known mutations.
Our system will be further validated by comparing phenotypes of the SNPs between two cell models, U2OS
and mPer2Luc MEFs.
Aim 2. Elucidate the underlying pathophysiology of critical mutations in CK1δ, CK1ε, Clock and Bmal1
genes. We will use our platform to test hypotheses on quantifiable changes previously proposed for specific
mutations in clock genes encoding CK1δ/ε, CLOCK and BMAL1. The majority of these mutations have not yet
been validated and quantified in in vivo models. We will focus on these mutations and dozens of SNPs at or
near these sites that could be as disruptive as the mutations identified by previous studies.
Aim 3. Develop a novel, specific treatment approach for circadian sleep disorders associated with
pathological SNPs . Current approaches to treat jet-lag or reset sleep cycles include light therapy, melatonin
and a few experimental drugs, none of which are specific to the clock and proven to be effective for circadian
disorders. We hypothesize that rapid degradation of limiting clock proteins using PROTACs can counteract the
pathogenicity of SNP mutations such that the clock is modulated to compensate the pathogenicity.
In summary, as we are now aware that human physiology is greatly impacted by defective clock mechanisms
associated with pathological SNPs in clock genes and CRISPR allows efficient genome editing, it is imperative
and timely to develop an efficient cell-based platform to study pathogenicity of these clinical SNPs.