Transcriptional feedback of Neurospora circadian clock gene by phosphorylation-dependent inactivation of its transcription factor

The circadian clock protein Frequency (FRO) feedback-regulates its own expression by inhibiting its transcriptional activator, White Collar Complex (WCC). We present evidence that FRO regulates the bulk of WCC through modulation of its phosphorylation status rather than via direct complex formation. In the absence of FRO, WCC is hypophosphorylated and transcriptionally active, while WCC is hyperphosphorylated and transcriptionally inactive when FRO is expressed. The phosphorylation status of WCC changes rhythmically over a circadian cycle. Dephosphorylation and activation of WCC depend on protein phosphatase 2A (PP2A), and WCC is a substrate of PP2A in vitro. Hypophosphorylated WCC binds to the clock box of the frq promoter even in the presence of FRO, while binding of hyperphosphorylated WCC is compromised even when FRO is depleted. We propose that negative feedback in the circadian clock of Neurospora is mediated by FRO, which rhythmically promotes phosphorylation of WCC, functionally equivalent to a cyclin recruiting cyclin-dependent kinase to its targets.