ChIP Assay for Protein-DNA Interactions

Chromatin immunoprecipitation (ChIP) experiments were performed as reported previously^{42 (link)}. Cells treated for 1 h in the high-light tolerance assay were harvested by centrifugation at 2000 × g at 4 °C for 2 min and resuspended in KH buffer (20 mM HEPES-KOH, pH7.6, and 80 mM KCl) containing 0.35% formaldehyde (Wako Chemical, Japan). After incubation for 10 min at room temperature, the cross-linking reaction was terminated with 1 M glycine for 5 min. The cross-linked cells were sonicated using the BIORUPTOR® II (Cosmo Bio, Japan) for 10 s (×30) with pauses of 20 s between each sonication cycle and then subjected to ChIP. The target DNA–protein complexes were immunoprecipitated using 100 μL of SURE-beads (Bio-Rad Laboratories) conjugated with 5 μg of FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich) at 4 °C for 1 h. After an overnight cross-link reversion between DNA and protein at 65 °C, the DNA was extracted and purified using the phenol/chloroform/isoamylalcohol purification method. The purified DNA was then subjected to semiquantitative PCR using KOD FX Neo DNA polymerase (TOYOBO) on the SimpliAmp Thermal Cycler (Thermo Fisher Scientific). Real-time qPCR assays were performed using the KOD SYBR® qPCR Mix (TOYOBO) on the Light Cycler 96 system (Roche Diagnostics, Germany). The primers used for PCR analyses are listed in Supplementary Table 7.