CRY2 missense mutations suppress P53 and enhance cell growth.

Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA-repair enzymes. While CRYs lack DNA-repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SKP1 cullin 1-F-box (SCF)FBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady-state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3 Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target-gene expression, suggesting that this may be a primary mechanism by which they influence cell growth.

The tail of cryptochromes: an intrinsically disordered cog within the mammalian circadian clock.

Cryptochrome (CRY) proteins play an essential role in regulating mammalian circadian rhythms. CRY is composed of a structured N-terminal domain known as the photolyase homology region (PHR), which is tethered to an intrinsically disordered C-terminal tail. The PHR domain is a critical hub for binding other circadian clock components such as CLOCK, BMAL1, PERIOD, or the ubiquitin ligases FBXL3 and FBXL21. While the isolated PHR domain is necessary and sufficient to generate circadian rhythms, removing or modifying the cryptochrome tails modulates the amplitude and/or periodicity of circadian rhythms, suggesting that they play important regulatory roles in the molecular circadian clock. In this commentary, we will discuss how recent studies of these intrinsically disordered tails are helping to establish a general and evolutionarily conserved model for CRY function, where the function of PHR domains is modulated by reversible interactions with their intrinsically disordered tails. Video abstract.

The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1.

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.