ROS1: Key Marker and Regulator of Accessible Chromatin in Arabidopsis [ATAC-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.

自ROS1作为首个真核DNA去甲基化酶（DNA demethylase）被发现以来已逾二十载，但其全基因组结合位点以及除主动DNA去甲基化之外的功能仍未明确。本研究借助先进的染色质免疫共沉淀测序（ChIP-seq）技术，发现ROS1可特异性结合几乎所有可及染色质区域，并在不同组织中与染色质可及性的动态变化呈显著关联，由此确立其作为可及染色质标记物的身份。此外，本研究证实ROS1可通过基于结合占据的机制维持DNA低甲基化状态，该机制能够阻碍RNA指导的DNA甲基化（RNA-directed DNA methylation, RdDM）的招募过程，这与其主动DNA去甲基化功能截然不同。除此之外，ROS1还可独立或与其他表观遗传调控因子协同作用，对染色质可及性发挥调控功能。该调控作用可发生于DNA甲基化依赖与非依赖两种情境中，ROS1作为可及染色质的潜在或实际保护因子，其功能取决于DNA甲基化系统的存在与靶向定位情况。本研究结果全面阐明了ROS1在染色质可及性与DNA甲基化调控中的功能，凸显了这两类调控机制之间复杂的串扰关系。实验整体设计：本研究利用课题组新近开发的aChIP技术，绘制了ROS1的全基因组结合图谱。我们发现ROS1可特异性结合几乎所有可及染色质区域，并在不同组织以及组蛋白去乙酰化酶（histone deacetylase）hda6突变体中与染色质可及性的动态变化呈显著关联，进而可作为可及染色质的标记物。通过整合ROS1与RNA指导的DNA甲基化（RdDM）结合占据数据，以及不同突变体与野生型植株的染色质可及性和DNA甲基化谱数据，本研究证实了ROS1在染色质可及性调控中的功能。此外，本研究提出了一种此前未被报道的调控机制：ROS1的结合占据可物理阻碍RdDM的招募，以此维持DNA低甲基化状态。本研究的发现阐明了这些调控机制之间复杂的串扰关系，为深入理解染色质结合蛋白的功能提供了全新的研究框架。