Defining a mechanism of circadian output in Neurospora crassa: temporal regulation of a MAP kinase pathway

Abstract

In the filamentous fungus Neuropora crassa, the Osmotically Sensitive (OS) signal transduction pathway is rhythmically activated and functions as an output pathway from the circadian clock. This conserved fungal pathway is composed of a sensory phosphorelay coupled to a mitogen activated protein kinase (MAPK) cascade. Our goal is to define the mechanism utilized by the FREQUENCY/WHITE COLLAR (FRQ/WCC) circadian oscillator to mediate rhythmicity of OS pathway activity that subsequently affects transcription of clock controlled genes. We show that expression of two components in the OS pathway, the histidine phosphotransferase encoded by hpt‐1 and the downstream MAPK kinase kinase encoded by os‐4, is regulated by the FRQ/WCC oscillator. We also demonstrate that the promoter of os‐4 is rhythmically bound by the White Collar Complex (WCC), a transcription factor and core component of the FRQ/WCC oscillator, with a period and phase that correlate with the rhythmic accumulation of os‐4 transcript. Furthermore, deletion of the promoter region bound by the WCC results in arrhythmic expression of os‐4 indicating that direct clock interaction is required for rhythmicity of os‐4. Interestingly, hpt‐1 expression rhythms peak in an opposite phase relative to os‐4 expression, and the hpt‐1 promoter is not bound directly by the WCC. Our research tests how clock regulation of OS pathway components affects the activation state of the pathway and expression of downstream genes. Our working model predicts that the activity of HPT‐1 inhibits pathway activation, while the activity of OS‐4 promotes pathway activation; therefore, by expressing these two components in opposite phases, the clock coordinates their activity to set the activation state of the pathway. By variably expressing the levels of these two components, we hypothesize that the clock can tune the basal level of pathway activation resulting in rhythmicity. This work is funded by a grant from the NIH.