Mechanistic Studies Of Mammalian CRYPTOCHROME Genes In Regulating Circadian Rhythms

Circadian clocks exist in most species on the earth. In mammals, the master clock locates in the suprachiasmatic nucleus (SCN), which is entrained by the light meanwhile synchronizes all the peripheral oscillators in the body. Indeed, circadian oscillations exist even at the single-cell level, behaving cell-autonomously. A widely accepted model for the intracellular clock is the transcription and translation feedback loop (TTFL). According to this model, the rhythmic expression of the clock-controlled genes (CCG) governs the output behaviors and physiology. Approximately 10% of the whole transcriptome is estimated to be under circadian regulations, including the clock genes themselves and clock-controlled genes.It has been demonstrated that the rhythmic expression of the positive regulators, such as BMAL1 and CLOCK, may not be necessary for the core oscillator to run. Conversely, the precise timing of rhythmic expression of negative regulators (PERIOD (PER1-3) and CRYPTOCHROME (CRY1/2)) is indispensable for proper clock functions. Although the PER and CRY complex has been well-established as a key component of the clock machinery, and the importance of PER proteins was most widely accepted especially at the post-translation level, the regulation of CRY proteins was less investigated in details.It is well known that both CRY1 and CRY2 determine the differences in the circadian period. However, the detailed mechanism underlying how CRYs regulate the length of the circadian period has not been well studied, although CRY protein stability is now widely accepted as an explanation for the circadian period regulation. In the present study, we are the first to show that CRY2, not only CRY1, is sufficient to restore circadian rhythm in CRY null cells. Although in contradiction with the protein stability hypothesis, we show that CRY2, which is more stable than CRY1, shorten the circadian period. The N-terminal of CRY2 contributes to the protein stability but appears to have little, if any, effect on the circadian period-length. Conversely, the most diverse C-terminal tail not only stabilizes the Photolyase homology region (PHR) domains but also regulates the nuclear importing rate of the protein, thereby determining the circadian circadian period length. Besides, we also found the interaction between CRY1 and P-TEFb complex/7SK snRNP, indicating the repression activity of CRY 1 through the 7SK snRNP.In summary, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period, more exactly, not PER or CRY individually, but the nuclear importing rate of PER complex determines the circadian period length. Our study also indicates the mammalian CRY executes the repression function through inactivating the P-TEFb complex. Altogether, our data provide new insight for the regulation of the circadian period and understanding the mechanism of CRY1 repression. Finally, we complete the circadian clock regulation loops.