Investigation Of The Molecular Mechanism Underlying The Regulation Of Neurospora Circadian Clock By Transcription Factor CBF-1

The circadian clock is of great physiological significance for the organism to predict the environment in advance and make changes so as to adapt to the environment better,improve efficiency and save energy.Neurospora crassa is one of the best-studied model systems for circadian rhythm.In the N.crassa circadian oscillator,WHITE COLLAR complex(WCC)actives frequency(frq)transcription.The accumulated FREQUENCY(FRQ)together with FRQ interaction RNA helicase(FRH)inhibits the activity of WCC to close the negative feedback loop.Rhythmic activation and repression of frq gene is essential for normal N.crassa circadian clock.Further exploration about the transcriptional repression is needed.I found that compared to WT,deletion of centromere binding factor(CBF-1)results in 2-hour longer period,lower amplitude and slower growth of circadian conidiation phenotype,which is dependent on its DNA binding ability.In the cbf-1KO,qa-Myc-CBF-1 strains,the leaky expression of Myc-CBF-1 without QA could restore the conidiation rhythm of cbf-1KO mutant.Addition a certain concentration of QA,however,results in a slow growth rate and arrhythmic conidiation rhythm.As expected,race tube assay showed that QA-induced the high levels of Myc-CBF-1 in the wt,qa-Myc-CBF-1 strain results in growth and circadian conidiation defects,suggesting that the proper level of CBF-1 is critical for circadian clock functions.Western blot and Northern blot showed that frq mRNA is increased in the cbf-1KO strains and frq mRNA is decreased when overexpressing CBF-1.So CBF-1 is a repressor for frq gene.However,we found that WCC activity is low in the cbf-1KO strains.Western blot and Northern blot showed that constant levels of frq mRNA and FRQ protein are observed in cbf-1KO wc-2KO double mutant.The phosphorylation levels of RCM-1 are increased and RCM-1 binding to C-box is reduced in the cbf-lKO strain,suggesting that CBF-1 suppresses WC-independent frq transcription by decreasing RCM-1 recruitment.EMSA and ChIP assays showed that CBF-1 rhythmically binds to the C-box region in vitro and in vivo.CBF-1 can suppress WCC binding at the C-box independent of FRQ.Overexpression of CBF-1 leads to increased CBF-1 at the C-box region but decreases WCC binding.The circadian conidiation phenotype is not affected by overexpression of these mutant Myc-CBF-1 proteins that cannot bind to frq C-box.ChIP and Western blot assays showed that the enrichment of WCC at the C-box and the levels of both WCC and FRQ are not affected in the wt,qa-Myc-CBF-lE195A or wt,qa-Myc-CBF-1?(187-214)strains.Together,these results suggest that CBF-1 binding to the C-box region impair WCC binding to its binding site in the C-box region.Based on above data,the mechanism is that the transcription factor CBF-1 acts as a repressor to suppress WC-independent frq transcription and modulate WCC for binding to the C-box of frq promoter.This study found that modulation of WCC binding at the frq promoter by CBF-1 ensures robust circadian oscillation of frq transcription,which indicates a new mechanism for circadian system.