Population epigenetics: DNA methylation in the plant omics era.

DNA methylation plays an important role in many biological processes. The mechanisms underlying the establishment and maintenance of DNA methylation are well understood thanks to decades of research using DNA methylation mutants, primarily in Arabidopsis (Arabidopsis thaliana) accession Col-0. Recent genome-wide association studies (GWASs) using the methylomes of natural accessions have uncovered a complex and distinct genetic basis of variation in DNA methylation at the population level. Sequencing following bisulfite treatment has served as an excellent method for quantifying DNA methylation. Unlike studies focusing on specific accessions with reference genomes, population-scale methylome research often requires an additional round of sequencing beyond obtaining genome assemblies or genetic variations from whole-genome sequencing data, which can be cost prohibitive. Here, we provide an overview of recently developed bisulfite-free methods for quantifying methylation and cost-effective approaches for the simultaneous detection of genetic and epigenetic information. We also discuss the plasticity of DNA methylation in a specific Arabidopsis accession, the contribution of DNA methylation to plant adaptation, and the genetic determinants of variation in DNA methylation in natural populations. The recently developed technology and knowledge will greatly benefit future studies in population epigenomes.

Epiallelic variation of non-coding RNA genes and their phenotypic consequences.

Epigenetic variations contribute greatly to the phenotypic plasticity and diversity. Current functional studies on epialleles have predominantly focused on protein-coding genes, leaving the epialleles of non-coding RNA (ncRNA) genes largely understudied. Here, we uncover abundant DNA methylation variations of ncRNA genes and their significant correlations with plant adaptation among 1001 natural Arabidopsis accessions. Through genome-wide association study (GWAS), we identify large numbers of methylation QTL (methylQTL) that are independent of known DNA methyltransferases and enriched in specific chromatin states. Proximal methylQTL closely located to ncRNA genes have a larger effect on DNA methylation than distal methylQTL. We ectopically tether a DNA methyltransferase MQ1v to miR157a by CRISPR-dCas9 and show de novo establishment of DNA methylation accompanied with decreased miR157a abundance and early flowering. These findings provide important insights into the genetic basis of epigenetic variations and highlight the contribution of epigenetic variations of ncRNA genes to plant phenotypes and diversity.

DNA conformational dynamics in the context-dependent non-CG CHH methylation by plant methyltransferase DRM2.

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.

Depositing centromere repeats induces heritable intragenic heterochromatin establishment and spreading in Arabidopsis.

Stable transmission of non-DNA-sequence-based epigenetic information contributes to heritable phenotypic variants and thus to biological diversity. While studies on spontaneous natural epigenome variants have revealed an association of epialleles with a wide range of biological traits in both plants and animals, the function, transmission mechanism, and stability of an epiallele over generations in a locus-specific manner remain poorly investigated. Here, we invented a DNA sequence deposition strategy to generate a locus-specific epiallele by depositing CEN180 satellite repeats into a euchromatic target locus in Arabidopsis. Using CRISPR/Cas9-mediated knock-in system, we demonstrated that depositing CEN180 repeats can induce heterochromatin nucleation accompanied by DNA methylation, H3K9me2, and changes in the nucleosome occupancy at the insertion sites. Interestingly, both DNA methylation and H3K9me2 are restricted within the depositing sites and depletion of an H3K9me2 demethylase IBM1 enables the outward heterochromatin propagation into the neighboring regions, leading to inheritable target gene silencing to persist for at least five generations. Together, these results demonstrate the promise of employing a cis-engineering system for the creation of stable and site-specific epialleles and provide important insights into functional epigenome studies and locus-specific transgenerational epigenetic inheritance.

Plant-specific histone deacetylases associate with ARGONAUTE4 to promote heterochromatin stabilization and plant heat tolerance.

High temperature causes devasting effects on many aspects of plant cells and thus enhancing plant heat tolerance is critical for crop production. Emerging studies have revealed the important roles of chromatin modifications in heat stress responses. However, how chromatin is regulated during heat stress remains unclear. We show that heat stress results in heterochromatin disruption coupled with histone hyperacetylation and DNA hypomethylation. Two plant-specific histone deacetylases HD2B and HD2C could promote DNA methylation and relieve the heat-induced heterochromatin decondensation. We noted that most DNA methylation regulated by HD2B and HD2C is lost upon heat stress. HD2B- and HD2C-regulated histone acetylation and DNA methylation are dispensable for heterochromatin maintenance under normal conditions, but critical for heterochromatin stabilization under heat stress. We further showed that HD2B and HD2C promoted DNA methylation through associating with ARGONAUTE4 in nucleoli and Cajal bodies, and facilitating its nuclear accumulation. Thus, HD2B and HD2C act both canonically and noncanonically to stabilize heterochromatin under heat stress. This study not only reveals a novel plant-specific crosstalk between histone deacetylases and key factor of DNA methylation pathway, but also uncovers their new roles in chromatic regulation of plant heat tolerance.

Structure and Mechanism of Plant DNA Methyltransferases.

DNA methylation is an important epigenetic mark conserved in eukaryotes from fungi to animals and plants, where it plays a crucial role in regulating gene expression and transposon silencing. Once the methylation mark is established by de novo DNA methyltransferases, specific regulatory mechanisms are required to maintain the methylation state during chromatin replication, both during meiosis and mitosis. Plant DNA methylation is found in three contexts; CG, CHG, and CHH (H = A, T, C), which are established and maintained by a unique set of DNA methyltransferases and are regulated by plant-specific pathways. DNA methylation in plants is often associated with other epigenetic modifications, such as noncoding RNA and histone modifications. This chapter focuses on the structure, function, and regulatory mechanism of plant DNA methyltransferases and their crosstalk with other epigenetic pathways.

Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana.

Histone post-translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a powerful tool to profile histone PTMs in vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)-based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high-quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high-yielding histone extraction and purification method optimized for Arabidopsis thaliana that can be used to obtain high-quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS-ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC-MS/MS and data analysis.

Mechanistic basis for maintenance of CHG DNA methylation in plants.

DNA methylation is an evolutionarily conserved epigenetic mechanism essential for transposon silencing and heterochromatin assembly. In plants, DNA methylation widely occurs in the CG, CHG, and CHH (H = A, C, or T) contexts, with the maintenance of CHG methylation mediated by CMT3 chromomethylase. However, how CMT3 interacts with the chromatin environment for faithful maintenance of CHG methylation is unclear. Here we report structure-function characterization of the H3K9me2-directed maintenance of CHG methylation by CMT3 and its Zea mays ortholog ZMET2. Base-specific interactions and DNA deformation coordinately underpin the substrate specificity of CMT3 and ZMET2, while a bivalent readout of H3K9me2 and H3K18 allosterically stimulates substrate binding. Disruption of the interaction with DNA or H3K9me2/H3K18 led to loss of CMT3/ZMET2 activity in vitro and impairment of genome-wide CHG methylation in vivo. Together, our study uncovers how the intricate interplay of CMT3, repressive histone marks, and DNA sequence mediates heterochromatic CHG methylation.

Linking transcriptional silencing with chromatin remodeling, folding, and positioning in the nucleus.

Chromatin organization is important for many DNA-templated processes in eukaryotic cells such as replication and transcription. Recent studies have uncovered the capacity of epigenetic modifications, phase separation, and nuclear architecture and spatial positioning to regulate chromatin organization in both plants and animals. Here, we provide an overview of the recent progress made in understanding how chromatin is organized within the nucleus at both the local and global levels with respect to the regulation of transcriptional silencing in plants. To be concise while covering important mechanisms across a range of scales, we focus on how epigenetic modifications and chromatin remodelers alter local chromatin structure, how liquid-liquid phase separation physically separates broader chromatin domains into distinct droplets, and how nuclear positioning affects global chromatin organization.

Quality control and evaluation of plant epigenomics data.

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.

BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity.

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.

DNA methyltransferase CHROMOMETHYLASE3 prevents ONSEN transposon silencing under heat stress.

DNA methylation plays crucial roles in transposon silencing and genome integrity. CHROMOMETHYLASE3 (CMT3) is a plant-specific DNA methyltransferase responsible for catalyzing DNA methylation at the CHG (H = A, T, C) context. Here, we identified a positive role of CMT3 in heat-induced activation of retrotransposon ONSEN. We found that the full transcription of ONSEN under heat stress requires CMT3. Interestingly, loss-of-function CMT3 mutation led to increased CHH methylation at ONSEN. The CHH methylation is mediated by CMT2, as evidenced by greatly reduced CHH methylation in cmt2 and cmt2 cmt3 mutants coupled with increased ONSEN transcription. Furthermore, we found more CMT2 binding at ONSEN chromatin in cmt3 compared to wild-type accompanied with an ectopic accumulation of H3K9me2 under heat stress, suggesting a collaborative role of H3K9me2 and CHH methylation in preventing heat-induced ONSEN activation. In summary, this study identifies a non-canonical role of CMT3 in preventing transposon silencing and provides new insights into how DNA methyltransferases regulate transcription under stress conditions.

Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions.

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

Substrate deformation regulates DRM2-mediated DNA methylation in plants.

DNA methylation is a major epigenetic mechanism critical for gene expression and genome stability. In plants, domains rearranged methyltransferase 2 (DRM2) preferentially mediates CHH (H = C, T, or A) methylation, a substrate specificity distinct from that of mammalian DNA methyltransferases. However, the underlying mechanism is unknown. Here, we report structure-function characterization of DRM2-mediated methylation. An arginine finger from the catalytic loop intercalates into the nontarget strand of DNA through the minor groove, inducing large DNA deformation that affects the substrate preference of DRM2. The target recognition domain stabilizes the enlarged major groove via shape complementarity rather than base-specific interactions, permitting substrate diversity. The engineered DRM2 C397R mutation introduces base-specific contacts with the +2-flanking guanine, thereby shifting the substrate specificity of DRM2 toward CHG DNA. Together, this study uncovers DNA deformation as a mechanism in regulating the specificity of DRM2 toward diverse CHH substrates and illustrates methylome complexity in plants.

Biological role and mechanism of chromatin readers in plants.

Epigenetic modifications are important gene regulatory mechanisms conserved in plants, animals, and fungi. Chromatin reader domains are protein-protein/DNA interaction modules acting within the chromatin-modifying complex to function as molecular interpreters of the epigenetic code. Understanding how reader proteins recognize specific epigenetic modifications and mediate downstream chromatin and transcriptional events is fundamental to many biological processes. Recent studies have uncovered a number of novel reader proteins with diverse functions and mechanisms in plants. Here, we provide an overview of the recent progress on reader-mark recognition modes, the mechanisms by which reader proteins influence chromatin dynamics, and how reader-chromatin interactions regulate biological function. Because of space limitations, this review focuses on reader domains in plants that specifically bind histone methylation, histone acetylation, and DNA methylation.

UVR8 interacts with de novo DNA methyltransferase and suppresses DNA methylation in Arabidopsis.

DNA methylation is an important epigenetic gene regulatory mechanism conserved in eukaryotes. Emerging evidence shows DNA methylation alterations in response to environmental cues. However, the mechanism of how cells sense these signals and reprogramme the methylation landscape is poorly understood. Here, we uncovered a connection between ultraviolet B (UVB) signalling and DNA methylation involving UVB photoreceptor (UV RESISTANCE LOCUS 8 (UVR8)) and a de novo DNA methyltransferase (DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2)) in Arabidopsis. We demonstrated that UVB acts through UVR8 to inhibit DRM2-mediated DNA methylation and transcriptional de-repression. Interestingly, DNA transposons with high DNA methylation are more sensitive to UVB irradiation. Mechanistically, UVR8 interacts with and negatively regulates DRM2 by preventing its chromatin association and inhibiting the methyltransferase activity. Collectively, this study identifies UVB as a potent inhibitor of DNA methylation and provides mechanistic insights into how signalling transduction cascades intertwine with chromatin to guide genome functions.

F-box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis.

DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), the de novo Arabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself. Here, we conducted yeast two-hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F-BOX KELCH 1 (CFK1), as a novel DRM2-interacting partner and targets DRM2 for degradation via the ubiquitin-26S proteasome pathway in Arabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS-seq) to determine the biological significance of CFK1-mediated DRM2 degradation. Loss-of-function CFK1 leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome-wide CHH hypomethylation and transcriptional de-repression at specific DRM2 target loci. This study uncovered a distinct mechanism regulating de novo DNA methyltransferase by CFK1 to control DNA methylation level.

Linking signaling pathways to histone acetylation dynamics in plants.

As sessile organisms, plants face versatile environmental challenges and require proper responses at multiple levels for survival. Epigenetic modification of DNA and histones is a conserved gene-regulatory mechanism and plays critical roles in diverse aspects of biological processes, ranging from genome defense and imprinting to development and physiology. In recent years, emerging studies have revealed the interplay between signaling transduction pathways, epigenetic modifications, and chromatin cascades. Specifically, histone acetylation and deacetylation dictate plant responses to environmental cues by modulating chromatin dynamics to regulate downstream gene expression as signaling outputs. In this review, we summarize current understandings of the link between plant signaling pathways and epigenetic modifications with a focus on histone acetylation and deacetylation.

An inferred fitness consequence map of the rice genome.

The extent to which sequence variation impacts plant fitness is poorly understood. High-resolution maps detailing the constraint acting on the genome, especially in regulatory sites, would be beneficial as functional annotation of noncoding sequences remains sparse. Here, we present a fitness consequence (fitCons) map for rice (Oryza sativa). We inferred fitCons scores (ρ) for 246 inferred genome classes derived from nine functional genomic and epigenomic datasets, including chromatin accessibility, messenger RNA/small RNA transcription, DNA methylation, histone modifications and engaged RNA polymerase activity. These were integrated with genome-wide polymorphism and divergence data from 1,477 rice accessions and 11 reference genome sequences in the Oryzeae. We found ρ to be multimodal, with ~9% of the rice genome falling into classes where more than half of the bases would probably have a fitness consequence if mutated. Around 2% of the rice genome showed evidence of weak negative selection, frequently at candidate regulatory sites, including a novel set of 1,000 potentially active enhancer elements. This fitCons map provides perspective on the evolutionary forces associated with genome diversity, aids in genome annotation and can guide crop breeding programs.