Celebrating the discovery of DNA demethylase

Maintaining correct DNA methylation patterns entails the addition of methyl groups by DNA methyltransferases and the active removal of methylation from DNA. Removing a methyl group from 5-methylcytosine requires breaking a strong C–C bond, suggesting that demethylation might occur by an alternative mechanism that does not involve severing this bond. Indeed, the discovery of the 5-methylcytosine DNA glycosylase (also known as DNA demethylase) REPRESSOR OF SILENCING 1 (ROS1) by (Gong et al., 2002) revolutionized thinking in this field, as the study of ROS1 revealed a mechanism by which 5-methylcytosine is excised and replaced by the DNA repair machinery. This special issue celebrates the 20th anniversary of the discovery of ROS1 and the remarkable research that followed. In the first review in this special issue, (Zhang et al., 2022) provide perspectives on the 20 years of research since the discovery of ROS1 and on what the next 20 years might hold. In the last 20 years, researchers have elucidated the biochemical pathway as well as regulatory mechanisms of active DNA demethylation by identifying key proteins, examining the functions of active DNA demethylation in regulating important processes such as development and stress responses, and exploring applications in agriculture and beyond. For example, linking the demethylation activity of ROS1 to the sequence-specific targeting of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) systems promises to generate powerful tools not only for basic research on site-specific epigenetic gene regulation, but also for applications such as creating epialleles for breeding and removing aberrant methylation in cancerous cells and aging cells for therapeutic purposes. Exciting results on the mechanisms of epigenetic regulation continue to emerge, particularly connecting DNA methylation with other epigenetic marks such as histone modifications. For example, a breakthrough report by (Liu, Chen, et al., 2022) details how a histone chaperone, nuclear autoantigenic sperm protein (NASP), recognizes histone H3. (Shang et al., 2022) reveal a mechanistic link between DNA methylation and histone methylation by Arabidopsis TRITHORAX histone methyltransferases, which are components of the COMPASS complex. In addition, ROS1 recruitment to specific loci remains an interesting question, and (Zhou et al., 2022) reveal a mechanism by which the gene silencing mark H3K9me2 regulates demethylation. Another major area of interest focuses on the epigenetic regulation of abiotic stress responses. A review by (Liu, Wang et al., 2022) explores the responses to salt, drought, heat, cold, nutrient, and ultraviolet light stresses, as well as to abscisic acid (ABA). In addition, they note interesting questions for future research, such as the need to understand how multiple epigenetic modifications interact, how transgenerational memory works, and how the epigenetic landscape differs in different cell types under specific stresses. In a research article, (Li et al., 2022) show that BRAHMA, a chromatin remodeler, recruits a histone deacetylase to modulate root system architecture in response to phosphate starvation in Arabidopsis. (Wang, Zuo, et al., 2022) reveal how global demethylation contributes to the enhanced cold tolerance in the polyploid plant trifoliate orange (Poncirus trifoliata) compared with its diploid progenitors. Moreover, the ABA signaling pathway has central functions in stress responses and new reports in this issue reveal how epigenetic regulation interacts with ABA signaling. (Wang et al., 2022) identify DEMETHYLATION REGULATOR 1 as a regulator of DNA methylation in the nuclear and mitochondrial genomes acting upstream of ROS1 and responding to ABA. In addition, (Tang et al., 2022) reveal how removal of the RNA modification N6-methyladenosine by an RNA demethylase modulates ABA responses. The epigenetic regulation of plant development continues to be a research focus. Germline development involves global reprogramming of DNA methylation patterns, and (He and Feng, 2022) review how this plays out in plant germline and vegetative cells (including the interactions between cell types and the functions of small interfering RNAs), how the regulation and dynamics of germline methylation differ in plants and mammals, and how DNA methylation helps defend the genome of germ cells from transposable elements. In another developmental study, (Ma et al., 2022) show that auxin acts through the methyltransferase SET DOMAIN GROUP 8 to stimulate the expression of key growth regulators for callus formation. In addition to understanding the roles of DNA methylation in development and stress responses, substantial progress has been made in understanding how DNA methylation affects genome structure. For example, (Zhou et al., 2022) review how R-loops, which comprise an RNA:DNA duplex and a displaced single strand of DNA, form and are resolved, and how they interact with other known epigenetic regulatory mechanisms including DNA, RNA, and histone modifications. In addition, (Wang et al., 2022) examine Hi-C data for rice mutants lacking a key methyltransferase and reveal key similarities between topologically associated domains in rice and Arabidopsis. Non-coding RNAs and transposable elements have key roles in epigenetic regulation and (Wang et al., 2023) review mechanisms involving small nucleolar RNAs and regulatory long non-coding RNAs, with a particular emphasis on their effects on agronomic traits. (Huang et al., 2023) use stringent criteria to re-annotate long microRNAs (lmiRNAs, 24 nucleotides in length) in rice, revealing that most lmiRNAs are derived from MITEs and providing insight on the evolution of lmiRNAs, their targets, and their regulatory mechanisms, particularly in stress responses. Intriguingly, research by (Wang et al., 2022) suggests that we have more to learn about epigenetic mechanisms, finding that activation of transposable elements induced by nitrogen starvation seems independently to occur against known epigenetic mechanisms such as DNA and histone methylation. The diverse studies since the discovery of ROS1 20 years ago have explored many areas of DNA methylation biology and have potential implications for improving crops in the face of major challenges posed by climate change and other factors, such as soil salinization. Moreover, as pointed out by (Zhang et al., 2022), research in this field faces challenges such as integrating the diverse epigenetic factors at play, including DNA modifications, histone modifications, non-coding RNAs, and chromatin remodeling. Future research will also need to address how epigenetic regulation contributes to the crosstalk between plant responses to different stresses, as well as to the balance between stress tolerance and crop yield. Therefore, the next 20 years of research in this field promises to be even more interesting as researchers incorporate new technologies and address current and emerging challenges in agriculture and human health.