Modulation of Circadian Gene Expression and Metabolic Compensation by the RCO-1 Corepressor of Neurospora crassa

Abstract

Neurospora crassa is a model organism for the study of circadian clocks, molecular machineries that confer ∼24-hr rhythms to different processes at the cellular and organismal levels. The FREQUENCY (FRQ) protein is a central component of the Neurospora core clock, a transcription/translation negative feedback loop that controls genome-wide rhythmic gene expression. A genetic screen aimed at determining new components involved in the latter process identified regulation of conidiation 1 (rco-1), the ortholog of the Saccharomyces cerevisiae Tup1 corepressor, as affecting period length. By employing bioluminescent transcriptional and translational fusion reporters, we evaluated frq and FRQ expression levels in the rco-1 mutant background observing that, in contrast to prior reports, frq and FRQ expression are robustly rhythmic in the absence of RCO-1, although both amplitude and period length of the core clock are affected. Moreover, we detected a defect in metabolic compensation, such that high-glucose concentrations in the medium result in a significant decrease in period when RCO-1 is absent. Proteins physically interacting with RCO-1 were identified through co-immunoprecipitation and mass spectrometry; these include several components involved in chromatin remodeling and transcription, some of which, when absent, lead to a slight change in period. In the aggregate, these results indicate a dual role for RCO-1: although it is not essential for core-clock function, it regulates proper period and amplitude of core-clock dynamics and is also required for the rhythmic regulation of several clock-controlled genes.

Transcription factor CBF-1 is critical for circadian gene expression by modulating WHITE COLLAR complex recruitment to the frq locus

“…Our results suggest that CBF-1 acts as a repressor for WC-independent frq transcription by promoting RCM-1 recruitment ( Fig 4F–4H ). WC-independent FRQ expression was previously observed in the rco-1 KO and rcm-1 RIP strains [ 27 , 28 , 46 ]. As shown here ( Fig 1A and 1B and S3 Fig ) and in a previous study [ 46 ], roles of CBF-1 and RCO-1/RCM-1 in control of the clock depend on conditions (i.e., glucose concentration and liquid/solid media).…”

Circadian Proteomic Analysis Uncovers Mechanisms of Post-Transcriptional Regulation in Metabolic Pathways

“…The global circadian repressor CSP-1 modulates the expression of ~800 genes whose products are predominantly involved in metabolic output (Lambreghts et al, 2009; Sancar et al, 2011) and has been implicated in the metabolic compensation mechanism that isolates the core clock from changes in glucose metabolism (Sancar et al, 2012; see, however, Olivares-Yañez et al, 2016). To examine the role of CSP-1 in regulating the metabolic proteome, we sampled fungal mats from a Δ csp-1 strain and prepared samples for TMT-MS analysis as done for the wildtype strain.…”

“…Recent work has identified other factors impacting cross-talk between metabolic state and circadian transcription (e.g., RCO-1 and RCM-1) and metabolic genes (e.g., CRE-1) (Cupertino et al, 2015; Olivares-Yañez et al, 2016). CRE-1 (NCU08807) was significantly rhythmic at the RNA and protein (p = 0.0013) level, demonstrating a metabolic-responsive transcription factor also regulated by the clock.…”

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

“…The observed damping might be caused by desynchronization of the single cells in the culture, as is the case of mammalian fibroblasts (Welsh et al, 2004). It is also possible that the experimental conditions, including media composition, are not optimal for rhythmicity in the species studied (Doherty and Kay, 2010;Hurley et al, 2014;Olivares-Yanez et al, 2016). The transcriptional rhythms in Nannochloropsis species could also be controlled by a damped oscillator.…”