Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression.
CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1(BMAL2) circadian transcriptional activation by CRY1-2.
Dardente
Hugues
H
Laboratory of Molecular Chronobiology, Douglas Hospital Research Centre, McGill University, Montreal, QC, Canada H4H 1R3.
Fortier
Erin E
EE
Martineau
Vincent
V
Cermakian
Nicolas
N
D056930:ARNTL Transcription Factors; D020816:Amino Acid Motifs; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001683:Biological Clocks; D056926:CLOCK Proteins; D019556:COS Cells; D002522:Chlorocebus aethiops; D056931:Cryptochromes; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D056931:Cryptochromes; D004247:DNA; D005420:Flavoproteins; D015534:Trans-Activators