The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

Circadian clocks are evolved to adapt to the daily environment changes under different conditions. The ability to maintain circadian clock functions in response to various stress and perturbations is important for organismal fitness. Here, we show that the nutrient sensing GCN2 signaling pathway is required for robust circadian clock function under amino acid starvation in Neurospora. The deletion of GCN2 pathway components disrupts rhythmic transcription of clock gene frq by suppressing WC complex binding at the frq promoter due to its reduced histone H3 acetylation levels. Under amino acid starvation, the activation of GCN2 kinase and its downstream transcription factor CPC-1 establish a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5. The arrhythmic phenotype of the GCN2 kinase mutants under amino acid starvation can be rescued by inhibiting histone deacetylation. Finally, genome-wide transcriptional analysis indicates that the GCN2 signaling pathway maintains robust rhythmic expression of metabolic genes under amino acid starvation. Together, these results uncover an essential role of GCN2 signaling pathway in maintaining robust circadian clock function in response to amino acid starvation and the importance of histone acetylation at the frq locus in rhythmic gene expression. For RNA-seq analyses, strains cultured in 2% glucose in constant light at 25 °C for 18h were havested. Total RNAs were extracted using Trizol reagents.The purity of the sample was determined by NanoPhotometer ® (IMPLEN, CA, USA)；The concentration and integrity of RNA samples were detected by Agilent 2100 RNA nano 6000 assay kit (Agilent Technologies, CA, USA). Sequencing libraries were generated using VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina ® (NR604-01/02) (Annoroad Gene Technology Co., Ltd, Beijing) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample.Libraries were prepared according to the manufacturer’s instructions and analyzed using 150bp paired-end Illumina sequencing (Annoroad Gene Technology, Beijing).