non-CG Methylation Research Articles

Non-CG DNA hypomethylation promotes photosynthesis and nitrogen fixation in soybean

Non-CG DNA methylation, a plant-specific epigenetic mark mainly regulated by chromomethylase (CMT), is known to play important roles in Arabidopsis thaliana. However, whether and to what extent non-CG DNA methylation modulates agronomic traits in crops remain to be explored. Here, we describe the consequences of non-CG DNA hypomethylation on development, seed composition, and yield in soybean (Glycine max). We created a Gmcmt mutant line lacking function of all four CMT genes. This line exhibited substantial hypomethylation of non-CG (CHG and CHH) sites. Non-CG hypomethylation enhanced chromatin accessibility and promoted or repressed the expression of hundreds of functionally relevant genes, including upregulation of GOLDEN-LIKE 10 (GmGLK10), which led to enhanced photosynthesis and, unexpectedly, improved nitrogen fixation efficiency. The Gmcmt line produced larger seeds with increased protein content. This study provides insights into the mechanisms of non-CG methylation-based epigenetic regulation of soybean development and suggests viable epigenetic strategies for improving soybean yield and nutritional value.

Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decrease global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins.

DNA methylation remodeling and the functional implication during male gametogenesis in rice

BackgroundEpigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure.ResultsHere we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization.ConclusionCollectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites.

We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5'-CG/5'-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (ΔA323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3' flanking position making it a CCA and CCC specific DNA methyltransferase.

Whole-genome methylation reveals tissue-specific differences in non-CG methylation in bovine

Whole-genome methylation reveals tissue-specific differences in non-CG methylation in bovine

Estimating genome-wide DNA methylation heterogeneity with methylation patterns

BackgroundIn a heterogeneous population of cells, individual cells can behave differently and respond variably to the environment. This cellular diversity can be assessed by measuring DNA methylation patterns. The loci with variable methylation patterns are informative of cellular heterogeneity and may serve as biomarkers of diseases and developmental progression. Cell-to-cell methylation heterogeneity can be evaluated through single-cell methylomes or computational techniques for pooled cells. However, the feasibility and performance of these approaches to precisely estimate methylation heterogeneity require further assessment.ResultsHere, we proposed model-based methods adopted from a mathematical framework originally from biodiversity, to estimate genome-wide DNA methylation heterogeneity. We evaluated the performance of our models and the existing methods with feature comparison, and tested on both synthetic datasets and real data. Overall, our methods have demonstrated advantages over others because of their better correlation with the actual heterogeneity. We also demonstrated that methylation heterogeneity offers an additional layer of biological information distinct from the conventional methylation level. In the case studies, we showed that distinct profiles of methylation heterogeneity in CG and non-CG methylation can predict the regulatory roles between genomic elements in Arabidopsis. This opens up a new direction for plant epigenomics. Finally, we demonstrated that our score might be able to identify loci in human cancer samples as putative biomarkers for early cancer detection.ConclusionsWe adopted the mathematical framework from biodiversity into three model-based methods for analyzing genome-wide DNA methylation heterogeneity to monitor cellular heterogeneity. Our methods, namely MeH, have been implemented, evaluated with existing methods, and are open to the research community.

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.

Domains Rearranged Methylase 2 maintains DNA methylation at large DNA hypomethylated shores and long-range chromatin interactions in rice.

DNA methylation plays an important role in gene regulation and genomic stability. However, large DNA hypomethylated regions known as DNA methylation valleys (DMVs) or canyons have also been suggested to serve unique regulatory functions, largely unknown in rice (Oryza sativa). Here, we describe the DMVs in rice seedlings, which were highly enriched with developmental and transcription regulatory genes. Further detailed analysis indicated that grand DMVs (gDMVs) might be derived from nuclear integrants of organelle DNA (NORGs). Furthermore, Domains Rearranged Methylase 2 (OsDRM2) maintained DNA methylation at short DMV (sDMV) shores. Epigenetic maps indicated that sDMVs were marked with H3K4me3 and/or H3K27me3, although the loss of DNA methylation had a negligible effect on histone modification within these regions. In addition, we constructed H3K27me3-associated interaction maps for homozygous T-DNA insertion mutant of the gene (osdrm2) and wild type (WT). From a global perspective, most (90%) compartments were stable between osdrm2 and WT plants. At a high resolution, we observed a dramatic loss of long-range chromatin loops in osdrm2, which suffered an extensive loss of non-CG (CHG and CHH, H = A, T, or C) methylation. From another viewpoint, the loss of non-CG methylation at sDMV shores in osdrm2 could disrupt H3K27me3-mediated chromatin interaction networks. Overall, our results demonstrated that DMVs are a key genomic feature in rice and are precisely regulated by epigenetic modifications, including DNA methylation and histone modifications. OsDRM2 maintained DNA methylation at sDMV shores, while OsDRM2 deficiency strongly affected three-dimensional (3D) genome architectures.

The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes.

Establishment of a proper DNA methylation landscape in mammalian oocytes is important for maternal imprinting and embryonic development. De novo DNA methylation in oocytes is mediated by the DNA methyltransferase DNMT3A, which has an ATRX-DNMT3-DNMT3L (ADD) domain that interacts with histone H3 tail unmethylated at lysine-4 (H3K4me0). The domain normally blocks the methyltransferase domain via intramolecular interaction and binding to histone H3K4me0 releases the autoinhibition. However, H3K4me0 is widespread in chromatin and the role of the ADD-histone interaction has not been studied in vivo. We herein show that amino-acid substitutions in the ADD domain of mouse DNMT3A cause dwarfism. Oocytes derived from homozygous females show mosaic loss of CG methylation and almost complete loss of non-CG methylation. Embryos derived from such oocytes die in mid-to-late gestation, with stochastic and often all-or-none-type CG-methylation loss at imprinting control regions and misexpression of the linked genes. The stochastic loss is a two-step process, with loss occurring in cleavage-stage embryos and regaining occurring after implantation. These results highlight an important role for the ADD domain in efficient, and likely processive, de novo CG methylation and pose a model for stochastic inheritance of epigenetic perturbations in germ cells to the next generation.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons

During postnatal development, the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This methylation is critical for transcriptional regulation, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). Here, we show in mice that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, an H3K36 methyltransferase mutated in NDD, is required for the patterning of megabase-scale H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes altered DNA methylation that overlaps with DNMT3A disorder models to drive convergent dysregulation of key neuronal genes that may underlie shared phenotypes in NSD1- and DNMT3A-associated NDDs. Our findings indicate that H3K36me2 deposited by NSD1 is important for neuronal non-CG DNA methylation and suggest that the H3K36me2-DNMT3A-non-CG-methylation pathway is likely disrupted in NSD1-associated NDDs.

DNA methylation signatures of duplicate gene evolution in angiosperms.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomics approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole genome duplicates were typically enriched for CG-only gene-body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was characteristic of more recent single-gene duplicates. Core angiosperm gene families differentiated into those which preferentially retain paralogs and 'duplication-resistant' families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

Hyper non-CG methylation of expanded plant disease resistance NLR genes

Key messageWe explored the phylogenomics and methylomics of NLR genes in 41 plant species and found that highly duplicated plant NLR genes are hyper methylated in non-CG context.

On the prediction of non-CG DNA methylation using machine learning.

DNA methylation can be detected and measured using sequencing instruments after sodium bisulfite conversion, but experiments can be expensive for large eukaryotic genomes. Sequencing nonuniformity and mapping biases can leave parts of the genome with low or no coverage, thus hampering the ability of obtaining DNA methylation levels for all cytosines. To address these limitations, several computational methods have been proposed that can predict DNA methylation from the DNA sequence around the cytosine or from the methylation level of nearby cytosines. However, most of these methods are entirely focused on CG methylation in humans and other mammals. In this work, we study, for the first time, the problem of predicting cytosine methylation for CG, CHG and CHH contexts on six plant species, either from the DNA primary sequence around the cytosine or from the methylation levels of neighboring cytosines. In this framework, we also study the cross-species prediction problem and the cross-context prediction problem (within the same species). Finally, we show that providing gene and repeat annotations allows existing classifiers to significantly improve their prediction accuracy. We introduce a new classifier called AMPS (annotation-based methylation prediction from sequence)that takes advantage of genomicannotations to achieve higher accuracy.

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Establishment and maintenance of DNA methylation in nematode feeding sites.

A growing body of evidence indicates that epigenetic mechanisms, particularly DNA methylation, play key regulatory roles in plant-nematode interactions. Nevertheless, the transcriptional activity of key genes mediating DNA methylation and active demethylation in the nematode feeding sites remains largely unknown. Here, we profiled the promoter activity of 12 genes involved in maintenance and de novo establishment of DNA methylation and active demethylation in the syncytia and galls induced respectively by the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita in Arabidopsis roots. The promoter activity assays revealed that expression of the CG-context methyltransferases is restricted to feeding site formation and development stages. Chromomethylase1 (CMT1), CMT2, and CMT3 and Domains Rearranged Methyltransferase2 (DRM2) and DRM3, which mediate non-CG methylation, showed similar and distinct expression patterns in the syncytia and galls at various time points. Notably, the promoters of various DNA demethylases were more active in galls as compared with the syncytia, particularly during the early stage of infection. Mutants impaired in CG or CHH methylation similarly enhanced plant susceptibility to H. schachtii and M. incognita, whereas mutants impaired in CHG methylation reduced plant susceptibility only to M. incognita. Interestingly, hypermethylated mutants defective in active DNA demethylation exhibited contrasting responses to infection by H. schachtii and M. incognita, a finding most likely associated with differential regulation of defense-related genes in these mutants upon nematode infection. Our results point to methylation-dependent mechanisms regulating plant responses to infection by cyst and root-knot nematodes.

MethylC-analyzer: a comprehensive downstream pipeline for the analysis of genome-wide DNA methylation

DNA methylation is a crucial epigenetic modification involved in multiple biological processes and diseases. Current approaches for measuring genome-wide DNA methylation via bisulfite sequencing (BS-seq) include whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and enzymatic methyl-seq (EM-seq). The computational analysis tools available for BS-seq data include customized aligners for mapping bisulfite-converted reads and computational pipelines for downstream data analysis. Current post-alignment methylation tools are specialized for the interpretation of CG methylation, which is known to dominate mammalian genomes, however, non-CG methylation (CHG and CHH, where H refers to A, C, or T) is commonly observed in plants and fungi and is closely associated with gene regulation, transposon silencing, and plant development. Thus, we have developed a MethylC-analyzer to analyze and visualize post-alignment WGBS, RRBS, and EM-seq data focusing on CG. The tool is able to also analyze non-CG sites to enhance deciphering genomes of plants and fungi. By processing aligned data and gene location files, MethylC-analyzer generates a genome-wide view of methylation levels and methylation in user-specified genomic regions. The meta-plot, for example, allows the investigation of DNA methylation within specific genomic elements. Moreover, our tool identifies differentially methylated regions (DMRs) and investigates the enrichment of genomic features associated with variable methylation. MethylC-analyzer functionality is not limited to specific genomes, and we demonstrated its performance on both plant and human BS-seq data. MethylC-analyzer is a Python- and R-based program designed to perform comprehensive downstream analyses of methylation data, providing an intuitive analysis platform for scientists unfamiliar with DNA methylation analysis. It is available as either a standalone version for command-line uses or a graphical user interface (GUI) and is publicly accessible at https://github.com/RitataLU/MethylC-analyzer.

Local diversity of drought resistance and resilience in Populus tomentosacorrelates with the variation of DNA methylation.

Little information is known about DNA methylation variation in shaping environment-specific drought resistance and resilience for tree adaptation. In this study, we leveraged RNA sequencing and whole-genome bisulfite sequencing data to dissect the distinction of epigenetic regulation under drought stress and rewater condition of Populus tomentosa accessions from three geographical regions. We demonstrated low resistance and high resilience for accessions from South. Non-CG methylation levels in promoter regions of Southern accessions were lower than accessions from higher latitudes and negatively regulated gene expression. CHH context methylation was more sensitive to drought stress, and the geographical-specific differentially methylated regions were scarcely changed by environmental fluctuation. We identified 60 conserved hub genes within the co-expression networks that correlate with photosynthetic and stomatal morphological traits. Epigenome-wide association studiesand genome-wide association studiesof these 60 hub genes revealed the interdependency between genetic and epigenetic variation in GATA9 and LECRK-VIII.2, whichwas associated with stomatal morphology and chlorophyll content. The natural epigenetic variation in GATA9 was also faithfully transmitted to progenies in two family-based F1 populations. This study indicates a functional relationship of DNA methylation diversity with drought resistance and resilience which offers new insights into plants' local adaptation to a stressful environment.

Parental methylation mediates how progeny respond to environments of parents and of progeny themselves.

Environments experienced by both parents and offspring influence progeny traits, but the epigenetic mechanisms that regulate the balance of parental vs. progeny control of progeny phenotypes are not known. We tested whether DNA methylation in parents and/or progeny mediates responses to environmental cues experienced in both generations. Using Arabidopsis thaliana, we manipulated parental and progeny DNA methylation both chemically, via 5-azacytidine, and genetically, via mutants of methyltransferase genes, then measured progeny germination responses to simulated canopy shade in parental and progeny generations. We first found that germination of offspring responded to parental but not seed demethylation. We further found that parental demethylation reversed the parental effect of canopy in seeds with low (Cvi-1) to intermediate (Col) dormancy, but it obliterated the parental effect in seeds with high dormancy (Cvi-0). Demethylation did so by either suppressing germination of seeds matured under white-light (Cvi-1) or under canopy (Cvi-0), or by increasing the germination of seeds matured under canopy (Col). Disruption of parental methylation also prevented seeds from responding to their own light environment in one genotype (Cvi-0, most dormant), but it enabled seeds to respond to their own environment in another genotype (Cvi-1, least dormant). Using mutant genotypes, we found that both CG and non-CG DNA methylation were involved in parental effects on seed germination. Parental methylation state influences seed germination more strongly than does the progeny's own methylation state, and it influences how seeds respond to environments of parents and progeny in a genotype-specific manner.

DNA methylation underpins the epigenomic landscape regulating genome transcription in Arabidopsis

BackgroundIt is challenging to determine the effect of DNA methylation on the epigenetic landscape and the function in higher organisms due to the lack of DNA methylation-free mutants.ResultsHere, the analysis of a recently generated Arabidopsis mutant completely devoid of DNA methylation reveals that DNA methylation underpins the genome-wide landscape of histone modifications. Complete loss of DNA methylation causes an upheaval of the histone modification landscape, including complete loss of H3K9me2 and widespread redistribution of active and H3K27me3 histone marks, mostly owing to the role of DNA methylation in initiating H3K9me2 deposition and excluding active marks and repressive mark H3K27me3; CG and non-CG methylation can act independently at some genomic regions while they act cooperatively at many other regions. The transcriptional reprogramming upon loss of all DNA methylation correlates with the extensive redistribution or switches of the examined histone modifications. Histone modifications retained or gained in the DNA methylation-free mutant serve as DNA methylation-independent transcriptional regulatory signals: active marks promote genome transcription, whereas the repressive mark H3K27me3 compensates for the lack of DNA hypermethylation/H3K9me2 at multiple transposon families.ConclusionsOur results show that an intact DNA methylome constitutes the scaffolding of the epigenomic landscape in Arabidopsis and is critical for controlled genome transcription and ultimately for proper growth and development.

Conditional GWAS of non-CG transposon methylation in Arabidopsis thaliana reveals major polymorphisms in five genes.

Genome-wide association studies (GWAS) have revealed that the striking natural variation for DNA CHH-methylation (mCHH; H is A, T, or C) of transposons has oligogenic architecture involving major alleles at a handful of known methylation regulators. Here we use a conditional GWAS approach to show that CHG-methylation (mCHG) has a similar genetic architecture-once mCHH is statistically controlled for. We identify five key trans-regulators that appear to modulate mCHG levels, and show that they interact with a previously identified modifier of mCHH in regulating natural transposon mobilization.