A Daily Cycle of White Collar Complex Dephosphorylation Sustains Circadian Rhythmicity in Neurospora

As a photoreceptor, the transcription factor complex WCC acutely activates ~5% of the genome in response to blue light, while as the circadian positive element in the dark WCC influences expression of about 40% of the transcriptome. Among WCC-regulated genes is frq which is both acutely light-activated through a pLRE and circadian-regulated through a C-box promoter element that is not active in constant light. The complex of FRQ, FRH, and CK1, the FFC, phosphorylates WCC at >95 sites, thereby repressing its activity and closing the circadian feedback loop in the dark. Although FFC has no described role in the light, we validated the expectation that FFC-driven WCC phosphorylation also silences C-box promoters in constant light, thereby confirming two classes of WCC targets, C-box-like that are normally repressed in the light and pLRE-like that remain light-active despite FFC-driven WCC phosphorylation. Genome-wide derepression of C-box-like promoters in frq-null fungi may explain reported non-circadian effects seen in some frq-null fungi including reduced virulence and conidiation. Reanalysis of WCC-mediated circadian activation and repression revealed that, while at dusk most WCC is phosphorylated and repressed, subsequent circadian activation is the result of transient dephosphorylation/derepression of just a small subset of this WCC pool; this small active pool drives expression of FRQ, nucleating the FFC, which rapidly re-phosphorylates the WCC pool to repress it, a phosphorylation/dephosphorylation cycle that can run for days without new WCC synthesis. The realization that both FFC and WCC are regulated primarily through phosphorylation rather than turnover leaves the circadian oscillator looking much like a phoscillator, emphasizing the primacy of post-translational regulation in timekeeping.