Characterization of the single nucleotide polymorphisms that effect repression activity and stability of cryptochrome2

The daily rhythm of sleep/wake cycles, body temperature, metabolism, hormone secretion and many other physiological activities are in control of the circadian mechanism which is conserved from simple cyanobacteria to complex humans. In mammals, the circadian clock is endogenous and highly regulated with complex transcriptional and translational feedback loops (TTFL). Entrainment of the rhythm through the whole body is maintained by suprachiasmatic nuclei (SCN) located in the anterior hypothalamus with the cues from the environment. Many physiological processes are linked to the circadian clock, and it is not surprising that disruption of the clock can lead to certain disorders as demonstrated in various studies. At the molecular circadian mechanism of mammals, Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and Muscle ARNT-Like-1 (BMAL1) heterodimerizes and transactivates the expression of clock controlled genes including Period (Per) and Cryptochrome (Cry) by binding to E-box(CACGTG). PER and CRY accumulate in the cytoplasm and translocate into the nucleus, where they repress BMAL1/CLOCK-driven transactivation. The inhibition of BMAL1/CLOCK is relieved by the breakdown of CRY and PER proteins, resetting the cycle. In this study, the effect of the rare CRY2 variants in the central clock mechanism is aimed to be investigated. To this end, the fifteen rare Cry2 variants are identified from the database of 1000 Genomes Project and Ensembl. The initial assessment of these fifteen variants is predicated to be pathogenic. Further experimental and structural studies lead us to study p.Pro123Leu CRY2, p.Ser210Ile CRY2, and p.Asp406His CRY2 variants, which are located at a functionally important domain of the CRY2 called the secondary pocket, plays a role in CLOCK binding. CRY2 variants were unable to repress BMAL1/CLOCK driven transcription. Further biochemical studies indicated these variants were less stable than the wild type CRY2. Additionally, compared to wild type CRY2 the p.Pro123Leu CRY2, the p.Asp406His CRY2 and p.Ser410Ile had reduced affinity to CLOCK assessed by co-immunoprecipitation. Finally, it has been observed that p.Ser210Ile CRY2 and p.Asp406His CRY2 are not capable of rescuing the circadian rhythm in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line by complementation test. Collectively our data suggest that the secondary pocket of CRY2 plays a significant role not only for the CLOCK binding but also for the stability and the proper circadian rhythm at the cellular level.