Role of RCO-1 in the control of circadian gene expression and metabolic compensation of the Neurospora crassa circadian clock.

Circadian clocks are endogenous molecular timekeepers, which organize physiology of organisms with respect to the external world, conferring daily rhythms to a large number of biological processes within the cell. These clocks are present in various organisms, impinging close to 24 hours rhythms in the regulation of gene expression, physiology and behavior. A circadian system can be conceptualized as composed of three parts: input mechanisms, a central oscillator and output pathways. Although a detailed molecular description of the core oscillator is available in model eukaryotes, there is limited information on the mechanisms that allows it to regulate rhythmic processes. Such "output pathways" are the least characterized aspect of circadian systems. The filamentous fungus Neurospora crassa has served for decades as a model organism for the study of circadian biology. In an effort to improve current knowledge of output pathways identifying new components involved in this process, a genetic screen was conducted in this fungus, and we identified potential regulatory candidates, among which we characterized the coKrepressor RCOK1 and its role in regulating circadian biology. RCOK1 is the orthologue of the Saccharomyces cerevisiae transcriptional coK repressor Tup1. Contrary to reports that emerged while developing this thesis, we provide evidence that RCOK1 is not an essencial core-clock component in Neurospora. We evaluated the status of the central clock observing that expression of the negative element of the core oscillator, frequency (frq), remains rhythmic in the absence of RCOK1. Interestingly, in this mutant both amplitude and period length of the oscillations were affected. Importantly, we observed a defect in metabolic compensation in Δrco-1, such that high Kglucose concentrations when RCOK1 is not present significantly decrease period. We identified several proteins involved in chromatin remodeling and TFs that physically interact with RCOK1 and, in fact, the absence of one of the latter group also leads to a change in period. In addition, the analysis of RCOK1 target genes revealed that it plays an important role for their circadian expression. In summary, these results indicate a dual role for RCOK1: although it is not essential for core-clock function, it regulates proper period and amplitude of frq-based expression, where as it is also required in output for the rhythmic regulation of several clock-controlled genes. In addition, in the need to develop sensitive methods to follow FRQ expression of Δrco-1 under solid-media culture conditions this thesis also contributed to resolve key aspects of the clockworks. Thus, with this same methodology, we molecularly confirmed FRQ data that was inferred from bioluminescence studies of a Δfwd-1 strain. Thus, we analyzed FRQ levels in a KO of the F-box protein FWD-1 (a ubiquitin ligase that mediates FRQ proteasomal degradation) and that therefore exhibits decreased FRQ turnover. With this, we provided critical evidence showing that circadian period determination does not relay on FRQ stability, but instead on its degree of posttranslational modifications. In toto, although this thesis had as a main objective to contribute defining output pathways components, not only it helped advancing the latter, but it also shed light on basic core-clock mechanisms related to period determination and also to metabolic compensation.