Investigation Of The Transcriptional Repressors For Neurospora Circadian Clock Gene Frequency

Circadian clock is advantageous for the adaption of an organism to its ambient environment, and Neurospora crassa is among one of the best model organisms for the study of circadian clock. Rhythmic activation and repression of clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, periodic transcription of the clock gene frequency(frq) is essential for the Neurospora circadian clock, which contains two phases:transcriptional activation and transcriptional repression. The transcription of the frequency (frq) gene is periodically activated by the WHITE COLLAR (WC) complex and suppressed by the FREQUENCY (FRQ) protein. Although we have known transcriptional activation of frq very well, little is known about the transcriptional repression offrq.Thus we proposed a hypothesis that a number of transcriptional repressors might regulate frq transcription. By a series of genetic screening, we found some candidates serving as such transcriptional repressor, such as the transcriptional co-repressor RCO-1. In rco-1mutants, both overt and molecular rhythms are abolished, frq mRNA levels are constantly high. Surprisingly, frq mRNA levels were constantly high in the rco-1we double mutants, indicating that RCO-1suppresses WC-independent transcription. Furthermore, RCO-1is required for maintaining normal chromatin structure at the frq locus. Deletion of H3K36methyltransferase set-2or the chromatin remodeling factor chd-1leads to WC-independent frq transcription and loss of overt rhythms. Our results further indicated that RCO-1acts together with SET-2and CHD-1to regulate normal chromatin structure at the frq locus that permits rhythmic frq transcription. In rco-1mutants, high level constitutive WC-independent FRQ promotes the phosphorylation of WC by recruiting CKⅠ and CKⅡ kinases, which results in impaired WC activity and the loss of circadian rhythmicity.To further investigate the regulation of the WC-independent frq transcription, we generated double mutants by crossing the we single mutants with some of the known Neurospora clock mutants. We found that mutation of the PKA regulatory subunit mcb results in WC-independent frq transcription. Moreover, elevated PKA activity also results in WC-independent frq transcription and the loss of clock function. We found that RCM-1is a phosphoprotein and is a substrate of PKA in vivo and in vitro. Protein purification experiments identified RCM-1as the protein partner of RCO-1and an essential component of the clock through its role in suppressing WC-independent frq transcription. Mutation of the PKA-dependent phosphorylation sites on RCM-1results in WC-independent transcription of frq and impaired clock function. Furthermore, we showed that RCM-1is associated with the chromatin at the frq locus, a process that is inhibited by PKA. Together, our results demonstrate that PKA regulates frq transcription by inhibiting RCM-1activity through RCM-1phosphorylation.This study not only uncovered a previously unexpected regulatory mechanism for clock gene transcription but also demonstrated that a highly conserved transcriptional repressor complex can regulate gene transcription by post-translational modifications. Our study will promote the understanding of the transcriptional regulation of the clock gene frq in Neurospora crassa, and may be instructive for related studies in other organisms.