Checkpoint kinases regulate the circadian clock after DNA damage by influencing chromatin dynamics [RNA-Seq]

Circadian rhythms allow organisms to adjust to daily environmental fluctuations. The interplay between the circadian clock, the cell cycle, and DNA repair has been extensively documented, yet the epigenetic control of the circadian clock by the DNA damage response pathway remains relatively unexplored. Here, we showed that checkpoint kinases CHK1 and CHK2 regulate chromatin structure under DNA damage stress in Neurospora crassa to maintain robust circadian rhythms. Under DNA damage stress, deletion of chk1 and chk2 disrupted the rhythmic transcription of the clock gene frq by suppressing the rhythmic binding of the transcription activator WCC at the frq promoter, as the chromatin structure remained condensed. Mechanistically, CHK1 and CHK2 interacted and bound at the frq promoter to phosphorylate H3T11, promoting H3 acetylation, especially H3K56 acetylation, to counteract the histone variant H2A.Z deposition, establishing a suitable chromatin state to maintain the robust circadian rhythm despite DNA damage. Additionally, a genome-wide correlation was discovered between H3T11 phosphorylation and H3K56 acetylation, showing a specific function at the frq promoter that is dependent on CHK1 and CHK2. Furthermore, transcriptome analysis revealed that CHK1 and CHK2 are responsible for robust rhythmic transcription of metabolic and DNA repair genes under DNA damage stress. These findings highlight the essential role of checkpoint kinases in maintaining robust circadian rhythms under DNA damage stress. Overall design: The WT and chk1KOchk2KO strains' mats were cut into discs and transferred into medium-containing flasks and were harvested at DD18. Total RNAs were extracted using Trizol reagents. Libraries were prepared according to the manufacturer's instructions and analyzed using 150 bp paired-end Illumina sequencing (Annoroad Gene Technology, Beijing). After sequencing, the raw data were treated and mapped to the genome of Neurospora crassa (GCF_000182925.2) and transformed into expression value. The gene-expression level was measured in TPM using StringTie v1.3.3. The differentially expressed genes were assessed using the DEseq2 v1.16.1 Bioconductor package and defined based on the fold change criterion (|log2fold change|) > 0.5,Padj< 0.1). The functional category enrichments, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms, were analyzed by TBtools, and visualized by R package ggplot2 v3.3.6.