Suppression of WC-independent frequency transcription by RCO-1 is essential for Neurospora circadian clock

Abstract

Rhythmic activation and repression of clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, frequency (frq) transcription requires the WHITE COLLAR (WC) complex. Here, we show that the transcriptional corepressor regulation of conidiation-1 (RCO-1) is essential for clock function by regulating frq transcription. In rco-1 mutants, both overt and molecular rhythms are abolished, frq mRNA levels are constantly high, and WC binding to the frq promoter is dramatically reduced. Surprisingly, frq mRNA levels were constantly high in the rco-1 wc double mutants, indicating that RCO-1 suppresses WC-independent transcription and promotes WC complex binding to the frq promoter. Furthermore, RCO-1 is required for maintaining normal chromatin structure at the frq locus. Deletion of H3K36 methyltransferase su(var)3-9-enhancerof-zeste-trithorax-2 (SET-2) or the chromatin remodeling factor CHD-1 leads to WC-independent frq transcription and loss of overt rhythms. Together, our results uncover a previously unexpected regulatory mechanism for clock gene transcription. E ukaryotic circadian oscillators are based on autoregulatory negative feedback loops, in which positive and negative elements drive rhythmic gene expression (1-4). Despite the evolutionary distance, the circadian oscillator mechanism of the filamentous fungus Neurospora crassa is very similar to the mechanisms of animals (5-7). In Neurospora, Drosophila, and mammals, the positive elements of the circadian negative feedback loops are heterodimeric transcriptional activation complexes formed by a pair of Per-Arnt-Sim (PAS) domaincontaining transcription factors that rhythmically activate the transcription of the negative elements.In the core Neurospora circadian negative feedback loop, the positive element is the heterodimeric WHITE COLLAR (WC) complex of WC-1 and WC-2 (8-15). The WC complex binds to the promoter elements of the frequency ( frq) gene and activates its transcription. To close the negative feedback loop, FRQ and its partner FRQ-interacting RNA helicase (FRH) form a complex that inhibits transcription of frq by mediating FRQ/FRH complex-dependent WC phosphorylation, a process that inhibits WC complex activity and promotes its cytoplasmic localization (13,14,(16)(17)(18)(19)(20)(21). FRQ is progressively phosphorylated by several kinases and degraded through the ubiquitin proteasome pathway (13,(22)(23)(24). After the degradation of FRQ protein, the WC complex is reactivated, and a new cycle begins. The rhythmic activation and repression of frq transcription generate rhythmic frq mRNA levels, which are the major basis of circadian gene expression.WC-1 and WC-2 are the only previously known transcription factors required for the frq transcription. In wc-1 or wc-2 mutants, the levels of frq mRNA and FRQ protein are extremely low (11), leading to the conclusion that WC complex is the major-and perhaps, the only-regulator of frq transcription. There are two WC complex binding sites on f...

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora

“…Our previous work has shown that there is WC-independent frq transcription in rco-1 KO mutants (1). Moreover, the constitutive transcription of WC-independent frq interferes with the negative feedback loop and disrupts the circadian oscillator in rco-1 KO mutants.…”

“…Chromatin Immunoprecipitation-ChIP assay was performed as described previously (1,25,36,38). Briefly, Neurospora tissues under the experimental conditions were fixed with 1% formaldehyde for 15 min at 25°C with shaking, the fixation reaction were stopped by adding 125 mM glycine with shaking for another 5 min.…”

“…Although frq transcription is WC-dependent, our previous studies have shown that frq can be transcribed in a WC-independent manner in the absence of transcriptional corepressors, RCO-1 or RCM-1 (1,25). Unlike the WC-dependent frq transcription, WC-independent frq transcription is constitutive, which disrupts the rhythmical WC-dependent frq transcription through interference the negative feedback loop.…”

“…Rhythmic activation and repression of frq transcription result in cyclic changes of frq mRNA abundance. In Neurospora, histone modifiers and chromatin remodelers are required for rhythmic transcription of frq gene and clock-controlled genes (1,9,(22)(23)(24). However, the mechanism of transcriptional regulation of frq by different histone modifications is not fully understood.…”

Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

“…However, we recently discovered that frq transcription can occur in a WC-independent manner, and the suppression of the WC-independent frq transcription by RCO-1 is essential for circadian clock function (29). The homolog of RCO-1 in Saccharomyces cerevisiae is Tup1, a component of the Tup1-Ssn6 complex that represses gene expression (30,31).…”