Checkpoint kinases regulate the circadian clock after DNA damage by influencing chromatin dynamics [ChIP-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: ChIP assays were performed as previously described. Cultures were grown in 300?ml 2 % glucose media containing HU at 25?. The samples were crosslinked with 1% formaldehyde and the crosslinking was quenched by glycine. Cultures were washed with PBS, ground with liquid nitrogen and lysed in lysis buffer (50?mM HEPES-NaOH, pH7.5, 1?mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% Na deoxycholate, 137 mM NaCl, 1?mM PMSF, 1?µg/mL leupeptin, 1?µg/mL pepstatin A). Chromatin was sonicated to an average size of ~500?bp and 1 mg protein was subjected to immunoprecipitation with 2 µL WC-2, 1 µL H3, 1 µL Pol II S2P (Abcam, ab5095), 4 µL H3T11ph, 1 µL H3K56ac (HUABIO, ET1608), 1 µL H3ac (Millipore, 06-599), 2 µL H2A.Z (30), c-Myc, and CHK-2 antibodies at 4?°C overnight. The beads were then washed successively with lysis buffer, lysis buffer + 0.5?M NaCl, buffer II (10?mM Tris, pH 8.0, 1?mM EDTA, 0.25?M LiCl, 1% NP-40, 1% Na deoxycholate) and buffer III (50?mM Tris, pH 8.0, 1?mM EDTA). The eluted DNA/protein complexes were treated at 65?°C overnight and then treated with 2?µL Proteinase K (10?µg/µL) to remove proteins at 45?°C for 1?hour. The DNA purified with ethanol precipitation. For ChIP-qPCR analysis, occupancies were normalized by the ratio of ChIP to Input DNA. The ChIP signals for H3ac, H3T11ph and H3K56ac were normalized to H3.