Forward and reverse genetic approaches to unveil novel regulators of the Neurospora crassa circadian clock

Circadian rhythms are a widespread phenomenon, present in almost all living organisms. These rhythms are controlled by molecular clocks that allow the coordination of metabolism, physiology, and behavior with day/night cycles. At the core of these clocks lies a topologically, albeit no phylogenetically, conserved transcriptional/translational circuitry capable of creating and sustaining daily oscillations, even under various external perturbations. Ancillary transcriptional feedback loops have been reported in various circadian model organisms, which provide increased resilience to environmental perturbations. In Neurospora crassa, one of the most studied circadian model systems, however, the participation of transcriptional components controlling the clockwork, in addition to those in the core clock, are largely unknown.

Here, we set out to identify novel transcriptional regulators in N. crassa that affect clock parameters, namely circadian period, using forward and reverse genetic approaches.

We first generated a collection of transcription factor (TF) knockout strains, each missing one of all known TFs in this fungus, expressing a clock-controlled luciferase reporter. Through this reverse-genetics approach, we identified 8 transcriptional regulators, previously associated with a broad range of biological processes, that are required for the proper functioning of the core clock. Serendipitously, we also identified and subsequently mapped a novel spontaneous single nucleotide missense mutation in the gene encoding casein kinase 1a, a kinase known to post-transcriptionally regulate core clock elements in different organisms, that leads to a hitherto undescribed period defect of over 6 h.

Our results provide new details for the understanding of transcriptional and posttranslational regulatory mechanisms influencing circadian period determination.

• Nombre: Felipe Muñoz Guzmán
• Laboratorio: Cronobiología
• Mención: Genética Molecular y Microbiología
• Director Tesis: Luis Larrondo