Diverse Roles of Arabidopsis ROS1 in Regulating Chromatin Accessibility and DNA Methylation [ChIP-seq]

Over two decades after the discovery of ROS1 as the first eukaryotic DNA demethylase, its genome-wide binding sites and functions beyond active DNA demethylation remain unknown. Here, using advanced ChIP-seq, we reveal that ROS1 specifically occupies nearly all accessible chromatin and dynamically correlates with changes in chromatin accessibility across tissues, establishing it as a marker of accessible chromatin. Furthermore, we demonstrate that ROS1 maintains DNA hypomethylation through an occupancy-based mechanism that prevents the recruitment of RNA-directed DNA methylation, distinct from its active DNA demethylation. Additionally, ROS1 plays a regulatory role in chromatin accessibility, both independently and in cooperation with other epigenetic regulators. This regulation occurs in both DNA methylation-dependent and independent contexts, with ROS1 functioning as a potential or actual protector of accessible chromatin, depending on the presence and targeting of DNA methylation systems. Our results provide a comprehensive understanding of the regulatory roles of ROS1 in chromatin accessibility and DNA methylation, highlighting the intricate crosstalk between these mechanisms. Overall design: We take advantage of our recently developed aChIP method to map the genome-wide binding landscape of ROS1. We reveal that ROS1 specifically occupies nearly all accessible chromatin regions and dynamically correlates with changes in chromatin accessibility across distinct tissues and in the hda6 histone deacetylase mutant, thereby marking accessible chromatin. By integrating ROS1 and RNA-directed DNA methylation (RdDM) occupancy data with chromatin accessibility and DNA methylation profiles from various mutants and wild-type plants, we demonstrate the regulatory role of ROS1 in chromatin accessibility. Additionally, we propose a previously unreported mechanism by which ROS1 occupancy physically prevents the recruitment of RdDM to maintain DNA hypomethylation. Our findings elucidate the intricate crosstalk among these mechanisms and provide a framework for a deeper understanding of chromatin protein functions.