A HAD family phosphatase CSP-6 regulates the circadian output pathway in Neurospora crassa

Abstract

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δ csp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δ whi-2 mutant phenotypes resemble Δ csp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δ csp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δ csp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.

Author summary

Though molecules and components in the core circadian oscillator are well studied in Neurospora, the mechanisms through which output pathways are coupled with core components are less well understood. In this study we investigated a HAD phosphatase, CSP-6; loss-of-function Δ csp-6 strains are overtly arrhythmic but have a functional core circadian oscillation. CSP-6 in association with WHI-2 dephosphorylates the core clock component WC-1 to regulate light-responses and development. To dissect the functions of CSP-6 in core clock and output, we screened known WC-1 targets and found that loss of CSP-6 causes misregulation of transcriptional/translational activation of ADV-1, a key regulator of output. Thus, loss of CSP-6-mediated dephosphorylation of WC-1 leads to loss of ADV-1 activation and is responsible for the complete loss of overt developmental rhythmicity in Δ csp-6.