Epigenetic regulation based on 6mA is Essential for Cold Stress Acclimation in Chlamydomonas reinhardtii

This study investigates the role of N6-methyladenine (6mA) DNA methylation in the cold stress response of Chlamydomonas reinhardtii, the model microalga. While cytosine methylation (5mC) has been extensively studied in various eukaryotes, the role of adenine methylation (6mA) in stress acclimation remains poorly understood, especially in microalgae. Utilizing a chemical approach, we inhibited de novo adenine methylation with pyrimidinedione, a known 6mA methyltransferase inhibitor, and assessed its effects under cold stress conditions (4 °C). Results demonstrated that while 5mC reduction did not significantly impact cell viability, inhibition of 6mA severely impaired the microalga's ability to acclimate to cold stress, ultimately leading to cell death. To elucidate the molecular mechanisms underlying this phenomenon, we employed Oxford Nanopore Technology (ONT) for genome-wide methylome profiling and Illumina RNA-seq for transcriptome analysis. Data integration revealed that 6mA methylation predominantly occurred near transcription start sites (TSS) and was correlated with the activation of stress-responsive genes critical for cold acclimation. Key regulatory elements, including members of the SnRK family, methyltransferases, and stress-associated enzymes, exhibited differential 6mA methylation patterns and gene expression profiles upon cold exposure. Inhibition of 6mA methylation disrupted these regulatory pathways, underscoring its essential role in gene activation during stress response. Our findings provide novel insights into the epigenetic regulation of stress acclimation in Chlamydomonas reinhardtii, highlighting the pivotal function of 6mA methylation in cold stress survival. This study not only advances our understanding of algal stress biology but also offers potential strategies for engineering microalgae for enhanced stress tolerance and biomolecule production.