Mutational scanning identified amino acids of the CLOCK exon 19-domain essential for circadian rhythms

Aim: In the mammalian circadian clock, the CLOCK/BMAL1 heterodimer binds to E-box enhancer elements in the promoters of its target genes to activate transcription. The classical Clock mice, the first circadian mouse mutant discovered, are behaviourally arrhythmic. In this mutant, CLOCK lacks a 51 amino acid domain corresponding to exon 19 (CLOCK Delta 19), which is required for normal transactivation. While the importance of this CLOCK domain for circadian rhythms is well established, the exact molecular mechanism is still unclear. Methods: Using CRISPR/Cas9 technology, we created a CLOCK knockout - CLOCK rescue system in human circadian reporter cells and performed systematic mutational scanning to assess the functionality of individual amino acids within the CLOCK exon 19-domain. Results: CLOCK knockout cells were arrhythmic, and circadian rhythms could be rescued by introducing wild-type CLOCK, but not CLOCK Delta 19. In addition, we identified several residues, whose mutation failed to rescue rhythms in CLOCK knockout cells. Many of these are part of the hydrophobic binding interface of the predicted dimer of the CLOCK exon 19-domain. Conclusion: Our data not only indicate that CLOCK/BMAL1 oligomerization mediated by the exon 19-domain is important for circadian dynamics but also suggest that the exon 19-domain provides a platform for binding coactivators and repressors, which in turn is required for normal circadian rhythms.