BZIP transcription factors and transcriptional regulatory networks in the neurospora circadian system

Circadian clocks are endogenous cellular timekeepers that confer daily rhythms to a large number of biological processes. These clocks are present in various organisms across different evolutionary lineages, in which they regulate close to 24-hours rhythms in gene expression, physiology and behavior, enabling individuals to anticipate predictable environmental variations. The ascomycete Neurospora crassa has played a key role in the unveiling of the molecular and genetic basis of these time-telling machineries. In Neurospora, as in other eukaryotes, the integration of a series of cellular and molecular processes gives rise to a robust cell-based pacemaker, capable of coordinating rhythmic control of several aspects of their biology. Although a detailed molecular description of the core oscillator or pacemaker is now possible in model eukaryotes, there is limited information on the mechanisms that allow it to regulate rhythmic processes. Such “output pathways”, the circuits through which the pacemaker endows different processes with rhythmicity, are the least characterized aspect of circadian systems. In Neurospora, a hierarchical arrangement of transcriptional regulators has been proposed as the main mechanism through which the clock regulates rhythmic gene expression.The different actors involved in such time relay, connecting the oscillator with overt rhythms, are however largely unknown. In addition, despite decades as a research model organism, little is known about transcriptional regulatory networks in this fungus and the vast majority of transcription factors in Neurospora remain uncharacterized. In an effort to improve current knowledge of output pathways, the most neglected aspect of circadian biology, in a clock model system and study transcriptional regulatory networks in a model eukaryote, we set out to characterize the bZIP family of transcriptional regulators in Neurospora, in the context of its circadian system. We report a complete revision of the list of sequence-specific DNA-binding proteins in this fungus, which resulted in the identification of several novel ones, including many bZIP proteins. As the few transcription factors that have been associated with output pathways in Neurospora have been shown to exhibit clock input, we evaluated whether the expression of bZIP encoding genes in this organism is under control of the circadian clock. By using a luciferase-based, high-throughput screening system, we identified several bZIP encoding genes whose expression is regulated by the circadian pacemaker. A major limitation in the study of transcriptional regulatory networks in Neurospora, such as those underlying clock regulated transcription, stems from the fact that little is known about the sequence preference of its transcription factors. With the goal of identifying and characterizing transcriptional regulatory networks in which the putative Neurospora transcription factors participate, we employed double-stranded DNA microarrays known as protein-binding microarrays, to determine the sequence preference of Neurospora transcription factors.Such an approach allows for rapid, high-throughput and unbiased characterization of the sequence specificity of DNA-binding proteins. This resulted in the determination of the sequence preference of over half of Neurospora predicted transcription factors, information that together with the various molecular tools available in Neurospora, led to the identification of a rhythmically expressed bZIP transcription factor, ADA-1, as a regulator of output pathways in Neurospora, controlling various output genes. In addition, this information allowed for the evaluation of the role of another bZIP transcription factor, ASL-1, in such pathways. Notably, this is the first report aimed at studying DNA-binding specificities on a global scale in the fungal kingdom outside of the yeast clade, representing a powerful resource for the study of transcriptional regulatory networks in filamentous fungi, the largest group within the fungal kingdom. Indeed, through the use of these data we identified, for the first time, a transcription factor that is required for growth under osmotic stress in Neurospora. Lastly, we report on the identification of a novel process involved in output pathways in Neurospora, namely cell fusion pathways, and we herein show it to be necessary for proper rhythms in a number of genes, including bZIP encoding genes. As a whole, the work reported in this Thesis, represents a major advancement in the study of bZIP proteins and transcriptional regulatory networks in Neurospora.