Search

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host., This work was supported by Spain Ministerio de Ciencia e Innovación—FEDER (Grant Nos. BFU2015-65037-P and AGL2016-79825-R to S.F.E. and G.G., respectively) and Generalitat Valenciana (Grant No. PROMETEU/2019/012 to S.F.E.). R.L.C. was supported by a fellowship from the Brazilian funding agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico Brasil, Grant No. 200052/2017-9) for the stay in València and from EMBO/EuropaBio (Grant No. 7548) for the stay in Cambridge.

Objectives Early life stress is known to have enduring biological effects, particularly with respect to health. Epigenetic modifications, such as DNA methylation, are a possible mechanism to mediate the biological effect of stress. We previously found correlations between maternal stress, newborn birthweight, and genome-wide measures of DNA methylation. Here we investigate ten genes related to the methylation/demethylation complex in order to better understand the impact of stress on health. Materials and methods DNA methylation and genetic variants at methylation/demethylation genes were assayed. Mean methylation measures were constructed for each gene and tested, in addition to genetic variants, for association with maternal stress measures based on interview and survey data (chronic stress and war trauma), maternal venous, and newborn cord genome-wide mean methylation (GMM), and birthweight. Results After cell type correction, we found multiple pairwise associations between war trauma, maternal GMM, maternal methylation at DNMT1, DNMT3A, TET3, and MBD2, and birthweight. Conclusions The association of maternal GMM and maternal methylation at DNMT1, DNMT3A, TET3, and MBD2 is consistent with the role of these genes in establishing, maintaining and altering genome-wide methylation patterns, in some cases in response to stress. DNMT1 produces one of the primary enzymes that reproduces methylation patterns during DNA replication. DNMT3A and TET3 have been implicated in genome-wide hypomethylation in response to glucocorticoid hormones. Although we cannot determine the directionality of the genic and genome-wide changes in methylation, our results suggest that altered methylation of specific methylation genes may be part of the molecular mechanism underlying the human biological response to stress.

DNA methylation was the first epigenetic modification to be detected in human cancers with specific relation to aberrant gene expression. Herein, DNA methylation analysis explains how epigenetic patterns affect gene expression level. Hypermethylation at tumor suppressor gene loci leads to increased tumorigenesis due to tumor suppressor gene silencing, whereas global hypomethylation of CpG islands (CGIs) is followed by genomic instability and aberrant activation of multiple oncogenes. Therefore, characterization of the genes which silenced or activated epigenetically in human tumor cells can improve our understanding of cancer biology. Different genome-wide methodologies are applied to evaluate methylation status. Various commonly conducted techniques for this evaluation are reviewed in this paper. We provided comparative description of the procedures, advantages, and drawbacks of genome-wide DNA methylation analysis methods and biological applications, to give information on selecting the appropriate method for different methylation studies.

Chinese hamster ovary (CHO) cells account for the production of the majority of biopharmaceutical molecules - however, the molecular basis for their versatile properties is not entirely understood yet and the underlying cellular processes need to be characterized in detail. One such process that is supposed to contribute significantly to CHO cell phenotype is methylation of DNA at cytosine residues. DNA methylation was shown to be involved in several central biological processes in humans and to contribute to diseases like cancer. Early studies of DNA methylation in CHO mostly focused on methylation of single recombinant genes and promoters and proved a correlation between DNA methylation status and recombinant gene expression or production stability. More recent publications utilized the CHO genomic and transcriptomic data available since 2011 and provided first insights into the CHO DNA methylation landscape and DNA methylation changes in response to effector molecules or culture conditions. Generally, further genome-wide studies of DNA methylation in CHO will be required to shed light on the relevance of this process regarding biopharmaceuticals production and might, e.g., address a potential link between CHO cell metabolism and DNA methylation or provide novel targets for rational cell line engineering.

Background There is increasing evidence for inter-individual methylation differences at CpG dinucleotides in the human genome, but the regional extent and function of these differences have not yet been studied in detail. For identifying regions of common methylation differences, we used whole genome bisulfite sequencing data of monocytes from five donors and a novel bioinformatic strategy. Results We identified 157 differentially methylated regions (DMRs) with four or more CpGs, almost none of which has been described before. The DMRs fall into different chromatin states, where methylation is inversely correlated with active, but not repressive histone marks. However, methylation is not correlated with the expression of associated genes. High-resolution single nucleotide polymorphism (SNP) genotyping of the five donors revealed evidence for a role of cis-acting genetic variation in establishing methylation patterns. To validate this finding in a larger cohort, we performed genome-wide association studies (GWAS) using SNP genotypes and 450k array methylation data from blood samples of 1128 individuals. Only 30/157 (19%) DMRs include at least one 450k CpG, which shows that these arrays miss a large proportion of DNA methylation variation. In most cases, the GWAS peak overlapped the CpG position, and these regions are enriched for CREB group, NF-1, Sp100 and CTCF binding motifs. In two cases, there was tentative evidence for a trans-effect by KRAB zinc finger proteins. Conclusions Allele-specific DNA methylation occurs in discrete chromosomal regions and is driven by genetic variation in cis and trans, but in general has little effect on gene expression. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0144-2) contains supplementary material, which is available to authorized users.

DNA methylation is a very important mechanism for regulating gene expression in normal development and cell differentiation. However, DNA methylation, when acting on the promoter region of a tumor suppressor gene, suppresses transcription and is one of the major epigenetic modifications involved in the development and progression of many cancers (Wajed et al., 2001; Morris and Chan 2015). In our laboratory, we have carried out methylation analysis of 14 different tumor suppressor genes in cell-free DNA from the peripheral blood of over 1400 cancer patients, and have detected more than a total of 1900 methylations. We performed an analysis of DNA methylations of 14 different tumor suppressor genes, by type of cancer, stage of cancer, function of tumor suppressor genes, and their correlation to genetic mutations.

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

Comprehensive studies have shown that DNA methylation plays vital roles in both loss of pluripotency and governance of the transcriptome during embryogenesis and subsequent developmental processes. Aberrant DNA methylation patterns have been widely observed in tumorigenesis, ageing and neurodegenerative diseases, highlighting the importance of a systematic understanding of DNA methylation and the dynamic changes of methylomes during disease onset and progression. Here we describe a facile and convenient approach for efficient targeted DNA methylation by fusing inactive Cas9 (dCas9) with an engineered prokaryotic DNA methyltransferase MQ1. Our study presents a rapid and efficient strategy to achieve locus-specific cytosine modifications in the genome without obvious impact on global methylation in 24 h. Finally, we demonstrate our tool can induce targeted CpG methylation in mice by zygote microinjection, thereby demonstrating its potential utility in early development., Understanding how DNA methylation regulates gene expression requires the capacity to deploy it to regions of interest. The authors generate a highly rapid and locus-specific CpG methylation tool by fusing dCas9 to MQ1 DNA methyltransferase and show efficacy at multiple sites in vitro and in vivo.

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

DNA methylation is an important epigenetic modification that contributes to the lineage commitment and specific functions of different cell types. In this study, we compared ENCODE-generated genome-wide DNA methylation profiles of human osteoblast with 21 other types of human cells in order to identify osteoblast-specific methylation events. For most of the cell strains, data from two isogenic replicates were included, resulting in a total of 51 DNA methylation datasets. We identified 852 significant osteoblast-specific differentially methylated CpGs (DMCs) and 295 significant differentially methylated regions (DMRs). Significant DMCs/DMRs were not enriched in CpG islands (CGIs) and promoters, but more strongly enriched in CGI shores/shelves and in gene body and intergenic regions. The genes associated with significant DMRs were highly enriched in biological processes related to transcriptional regulation and critical for regulating bone metabolism and skeletal development under physiologic and pathologic conditions. By integrating the DMR data with the extensive gene expression and chromatin epigenomics data, we observed complex, context-dependent relationships between DNA methylation, chromatin states, and gene expression, suggesting diverse DNA methylation-mediated regulatory mechanisms. Our results also highlighted a number of novel osteoblast-relevant genes. For example, the integrated evidences from DMR analysis, histone modification and RNA-seq data strongly support that there is a novel isoform of neurexin-2 (NRXN2) gene specifically expressed in osteoblast. NRXN2 was known to function as a cell adhesion molecule in the vertebrate nervous system, but its functional role in bone is completely unknown and thus worth further investigation. In summary, we reported a comprehensive analysis of osteoblast-specific DNA methylation profiles and revealed novel insights into the epigenetic basis of osteoblast differentiation and activity., Highlights • Identify and characterize osteoblast-specific methylation patterns across the genome • Osteoblastic-specific methylation enriched in regulatory regions beyond the promoters • Diverse, context-dependent DNA methylation-mediated regulatory mechanisms • Revealed novel insights into the epigenetic basis of osteoblast differentiation

Despite numerous studies done on understanding the role of DNA methylation, limited work has focused on systems integration of cell type-specific interplay between DNA methylation and gene transcription. Through a genome-wide analysis of DNA methylation across 19 cell types with T-47D as reference, we identified 106,252 cell type-specific differentially-methylated CpGs categorized into 7,537 differentially (46.6% hyper- and 53.4% hypo-) methylated regions. We found 44% promoter regions and 75% CpG islands were T-47D cell type-specific methylated. Pyrosequencing experiments validated the cell type-specific methylation across three benchmark cell lines. Interestingly, these DMRs overlapped with 1,145 known tumor suppressor genes. We then developed a Bayesian Gaussian Regression model to measure the relationship among DNA methylation, genomic segment distribution, differential gene expression and tumor suppressor gene status. The model uncovered that 3′UTR methylation has much less impact on transcriptional activity than other regions. Integration of DNA methylation and 82 transcription factor binding information across the 19 cell types suggested diverse interplay patterns between the two regulators. Our integrative analysis reveals cell type-specific and genomic region-dependent regulatory patterns and provides a perspective for integrating hundreds of various omics-seq data together.

Eukaryotic genomes contain numerous non-functional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences similar to the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. In this study, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescence-labeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (∼10-30-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in non-functional locations. This effect may occur in vivo for DNA methylation outside CpG islands (CGIs) and could facilitate localization of some transcription factors within regulatory CGIs, where DNA methylation is rare.

Background DNA methylation is an important epigenetic mechanism involved in gene regulation, with alterations in DNA methylation in the nuclear genome being linked to numerous complex diseases. Mitochondrial DNA methylation is a phenomenon that is receiving ever-increasing interest, particularly in diseases characterized by mitochondrial dysfunction; however, most studies have been limited to the investigation of specific target regions. Analyses spanning the entire mitochondrial genome have been limited, potentially due to the amount of input DNA required. Further, mitochondrial genetic studies have been previously confounded by nuclear-mitochondrial pseudogenes. Methylated DNA Immunoprecipitation Sequencing is a technique widely used to profile DNA methylation across the nuclear genome; however, reads mapped to mitochondrial DNA are often discarded. Here, we have developed an approach to control for nuclear-mitochondrial pseudogenes within Methylated DNA Immunoprecipitation Sequencing data. We highlight the utility of this approach in identifying differences in mitochondrial DNA methylation across regions of the human brain and pre-mortem blood. Results We were able to correlate mitochondrial DNA methylation patterns between the cortex, cerebellum and blood. We identified 74 nominally significant differentially methylated regions (p

DNA methylation and demethylation is part of the essential biological processes regulating gene expression in normal cell development. Abnormal methylation status of specific genes and their irregularly translated proteins are normally associated with certain kinds of diseases or cancer. The rapid development of innovative DNA methylation mapping techniques provides a better understanding of DNA methylation pattern and its mechanisms in the human genome and its correlation with numerous diseases. These new techniques can lead us to develop new epigenetic medications, such as DNA methyltransferase inhibitors. As part of the approaches to probe DNA methylation and evaluate the effects of epigenetic therapy, mass spectrometry has been taking an important role in the identification, validation, and quantification of DNA methylation and demethylation. In this review, we will briefly summarize the current breadth of knowledge on the topic of DNA methylation and its occurrence in diseases, DNA methylation drugs, and mass spectrometry based approaches used to study DNA methylation.

Chemical modification of nucleobases plays an important role for the control of gene expression on different levels. That includes the modulation of translation by modified tRNA-bases or silencing and reactivation of genes by methylation and demethylation of cytosine in promoter regions. Especially dynamic methylation of adenine and cytosine is essential for cells to adapt to their environment or for the development of complex organisms from a single cell. Errors in the cytosine methylation pattern are associated with most types of cancer and bacteria use methylated nucleobases to resist antibiotics. This Point of View wants to shed light on the known and potential chemistry of DNA and RNA methylation and demethylation. Understanding the chemistry of these processes on a molecular level is the first step towards a deeper knowledge about their regulation and function and will help us to find ways how nucleobase methylation can be manipulated to treat diseases.

Cytosine DNA methylation is a vital epigenetic regulator of eukaryotic development. Whether this epigenetic modification occurs in Tribolium castaneum has been controversial, its distribution pattern and functions have not been established. Here, using bisulphite sequencing (BS-Seq), we confirmed the existence of DNA methylation and described the methylation profiles of the four life stages of T. castaneum. In the T. castaneum genome, both symmetrical CpG and non-CpG methylcytosines were observed. Symmetrical CpG methylation, which was catalysed by DNMT1 and occupied a small part in T. castaneum methylome, was primarily enriched in gene bodies and was positively correlated with gene expression levels. Asymmetrical non-CpG methylation, which was predominant in the methylome, was strongly concentrated in intergenic regions and introns but absent from exons. Gene body methylation was negatively correlated with gene expression levels. The distribution pattern and functions of this type of methylation were similar only to the methylome of Drosophila melanogaster, which further supports the existence of a novel methyltransferase in the two species responsible for this type of methylation. This first life-cycle methylome of T. castaneum reveals a novel and unique methylation pattern, which will contribute to the further understanding of the variety and functions of DNA methylation in eukaryotes.

Background DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. Results The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Conclusions Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0125-5) contains supplementary material, which is available to authorized users.

In humans, DNA methylation at specific CpG sites can be used to estimate the ‘epigenetic clock’, a biomarker of aging and health. The mechanisms that regulate the aging epigenome and level of conservation are not entirely clear. We performed affinity-based enrichment with methyl-CpG binding domain protein followed by high-throughput sequencing (MBD-seq) to assay DNA methylation in mouse samples. Consistent with previous reports, aging is associated with increase in methylation at CpG islands that likely overlap regulatory regions of genes that have been implicated in cancers (e.g., C1ql3, Srd5a2 and Ptk7). The differentially methylated regions in mice have high sequence conservation in humans and the pattern of methylation is also largely conserved between the two species. Based on human ENCODE data, these sites are targeted by polycomb proteins, including EZH2. Chromatin immunoprecipitation confirmed that these regions interact with EZH2 in mice as well, and there may be reduction in EZH2 occupancy with age at C1ql3. This adds to the growing evidence that EZH2 is part of the protein machinery that shapes the aging epigenome. The conservation in both sequence and methylation patterns of the age-dependent CpGs indicate that the epigenetic clock is a fundamental feature of aging in mammals.

Obesity is a highly prevalent, chronic disorder that has been increasing in incidence in young patients. Both epigenetic and genetic aberrations may play a role in the pathogenesis of obesity. Therefore, in-depth epigenomic and genomic analyses will advance our understanding of the detailed molecular mechanisms underlying obesity and aid in the selection of potential biomarkers for obesity in youth. Here, we performed microarray-based DNA methylation and gene expression profiling of peripheral white blood cells obtained from six young, obese individuals and six healthy controls. We observed that the hierarchical clustering of DNA methylation, but not gene expression, clearly segregates the obese individuals from the controls, suggesting that the metabolic disturbance that occurs as a result of obesity at a young age may affect the DNA methylation of peripheral blood cells without accompanying transcriptional changes. To examine the genome-wide differences in the DNA methylation profiles of young obese and control individuals, we identified differentially methylated CpG sites and investigated their genomic and epigenomic contexts. The aberrant DNA methylation patterns in obese individuals can be summarized as relative gains and losses of DNA methylation in gene promoters and gene bodies, respectively. We also observed that the CpG islands of obese individuals are more susceptible to DNA methylation compared to controls. Our pilot study suggests that the genome-wide aberrant DNA methylation patterns of obese individuals may advance not only our understanding of the epigenomic pathogenesis but also early screening of obesity in youth.

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

This review describes how disruptor of telomeric silencing 1-like (DOT1L) and histone H3 at lysine 79 (H3K79) methylation maintain genome stability under normal conditions and discusses the consequences of their dysregulation. We highlight the roles that DOT1L and H3K79 methylation have in various cellular processes and detail their roles in the DNA damage response and mitotic fidelity. DOT1L is currently the only known enzyme capable of catalyzing the methylation of H3K79. DOT1L activity and H3K79 methylation regulate a number of key cellular processes required to maintain genome stability, including transcription, cell cycle progression, and the DNA damage response. Consequently, alterations of DOT1L activity and H3K79 methylation patterning are predicted to compromise genome stability. Consistent with a putative role in oncogenesis, aberrant DOT1L expression and function occur in a wide range of cancer types including breast, colorectal, and lung, yet the exact mechanisms linking altered DOT1L expression and H3K79 methylation to genome instability remain poorly understood.

Imprinting is an epigenetic phenomenon in which genes are expressed selectively from either the maternal or paternal alleles. In plants, imprinted gene expression is found in a tissue called the endosperm. Imprinting is often set by a unique epigenomic configuration in which the maternal chromosomes are less DNA methylated than their paternal counterparts. In this review, we synthesize studies that paint a detailed molecular portrait of the distinctive endosperm methylome. We will also discuss the molecular machinery that shapes and modifies this methylome, and the role of DNA methylation in imprinting. Keywords: Gene imprinting; DNA methylation; plants; seeds; endosperm, National Science Foundation (U.S.) (Grant 1453459)

DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

There is substantial evidence of impaired DNA repair activities in Alzheimer's disease (AD) neurons and peripheral tissues, inducing some investigators to speculate that this could partially result from promoter hypermethylation of DNA repair genes, resulting in gene silencing in those tissues. In the present study a screening cohort composed by late-onset AD (LOAD) patients and healthy matched controls was evaluated with a commercially available DNA methylation array for the assessment of the methylation levels of a panel of 22 genes involved in major DNA repair pathways in blood DNA. We then applied a cost-effective PCR based methylation-sensitive high-resolution melting (MS-HRM) technique, in order to evaluate the promoter methylation levels of the following DNA repair genes: OGG1, PARP1, MRE11A, BRCA1, MLH1, and MGMT. The analysis was performed in blood DNA from 56 LOAD patients and 55 matched controls, including the samples previously assessed with the DNA methylation array as validating samples. Both approaches revealed that all the investigated genes were largely hypomethylated in LOAD and control blood DNA, and no difference between groups was observed. Collectively, present data do not support an increased promoter methylation of some of the major DNA repair genes in blood DNA of AD patients.

In mammals, DNA methylation is a crucial epigenetic modification with key functions during development. Cellular processes that are regulated by DNA methylation comprise X chromosome inactivation, gene imprinting, genomic stability and transcriptional regulation. Generally, the methylation status of the majority of target sites is reliably propagated during mitosis. However, advances in genome-wide DNA methylation analysis at base-resolution have discovered a substantial amount of differential DNA methylation between normal cells of different tissue-origin. Moreover, aberrant DNA methylation changes are linked with a significant number of human diseases, particularly with cancer. Sites of differential and aberrant DNA methylation include regulatory DNA sequences, such as CpG islands in promoters and distal cis-regulatory elements, like enhancers. In this review, we will discuss novel aspects of DNA methylation dynamics, during normal development and in association with diseases.

Background/Aims: This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. Methods: To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. Results: The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P

Plant DNA methylation is its own language, interpreted by the cell to maintain silencing of transposons, facilitate chromatin structure, and to ensure proper expression of some genes. Just as in any language, context is important. Rather than being a simple "on-off switch", DNA methylation has a range of "meanings" dependent upon the underlying sequence and its location in the genome. Differences in the sequence context of individual sites are established, maintained, and interpreted by differing molecular pathways. Varying patterns of methylation within genes and surrounding sequences are associated with a continuous range of expression differences, from silencing to constitutive expression. These often-subtle differences have been pieced together from years of effort, but have taken off with the advent of methods for assessing methylation across entire genomes. Recognizing these patterns and identifying underlying causes is essential for understanding the function of DNA methylation and its systems-wide contribution to a range of processes in plant genomes. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1–AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.

Epigenetic modifications are responsible for the modulation of gene expression without affecting the nucleotide sequence. The observed changes in transcriptional activity of genes in tumor tissue compared to normal tissue, are often the result of DNA methylation within the promoter sequences of these genes. This modification by attaching methyl groups to cytosines within CpG islands results in silencing of transcriptional activity of the gene, which in the case of tumor suppressor genes is manifested by abnormal cell cycle, proliferation and excessive destabilization of the repair processes. Further studies of epigenetic modifications will allow a better understanding of mechanisms of their action, including the interdependence between DNA methylation and activity of proteins crucial to the structure of chromatin and gene activity. Wider knowledge of epigenetic mechanisms involved in the process of malignant transformation and pharmacological regulation of the degree of DNA methylation provides an opportunity to improve the therapeutic actions in the fight against cancer.

DNA Methylation is an epigenetic phenomenon in which methyl groups are added to the cytosines, thereby altering the physio-chemical properties of the DNA region and influencing gene expression. Aberrant DNA methylation in a set of genes or across the genome results in many epigenetic diseases including cancer. In this paper, we use entropy to analyze the extent and distribution of DNA methylation in Tumor Suppressor Genes (TSG's) and Oncogenes related to a specific type of cancer (viz.) KIRC (Kidney-renal-clear-cell-carcinoma). We apply various mathematical transformations to enhance the different regions in DNA methylation distribution and compare the resultant entropies for healthy and tumor samples. We also obtain the sensitivity and specificity of classification for the different mathematical transformations. Our findings show that it is not just the measure of methylation, but the distribution of the methylation levels in the genes that are significant in cancer.

Colorectal cancer (CRC) is one of the most common and severe cancers worldwide. The occurrence of CRC is developed by accumulation of genetic and epigenetic alteration in colon cells. Work over the last decade has proposed that epigenetic changes such as DNA methylation, histone modification of protein coding genes play an important role in CRC development. However, the epigenetic pattern and features of lncRNAs in CRC were unclear. Here, we comprehensively analyze the patterns of DNA methylation, H3K4me3, H3K27me3 on both protein coding genes and lncRNAs. We found several interesting results which may help to discriminate the lncRNAs from protein coding genes. For example, the signals of DNA methylation and H3K4me3 are higher on protein coding genes than lncRNAs, but not for H3K27me3; the three epigenetic marks show different distribution on promoters, termination and across the whole gene between protein coding genes and lncRNAs, especially DNA methylation, which show regular signal tendency according to the principle of gene transcription. In addition, we further analyzed the affections of epigenetic marks on protein coding gene and lncRNA expression in HCT116 colon cell. Most of the results were consistent with the previous reports such as H3K27me3 is an repressive mark. Furthermore, we analyzed the relationships among the three epigenetic marks and found that DNA methylation and H3K4me3 were positively correlated in promoter and termination region for both protein coding genes and lncRNAs. In a word, our results will give a clue to further study the pathologies of CRC.

Little is known about the contributions of DNA methylation/demethylation to plant aging and senescence. We used Arabidopsis thaliana to study how increasing age of an annual plant species influences DNA methylation. Based on methylation-sensitive DNA fragmentation assay, it could be concluded that aging A. thaliana was accompanied by DNA demethylation. Bisulfite sequencing reveals that cytosine methylation within the Actin2 3’ untranslated region and internal transcribed spacer with 5.8S rRNA (ITS1-5.8SrRNA-ITS2) DNA regions decreased with A. thaliana growth and aging. We show that transcription of methyltransferase genes, chromomethyltransferase AtCMT3 and methyltransferse AtMETI, significantly decreased during development and aging of the A. thaliana plants, whereas expression of demethylase genes - repressor of silencing AtROS1, demeter AtDME, and demeter-like AtDML2 and AtDML3 - increased at least at some stages of plant development. The data obtained in the present study suggest that plant DNA regions may undergo demethylation during plant aging via reduction of DNA methylation processes and activation of active DNA demethylation.

DNA methylation is one of the epigenetic marks and plays critically important functions during spermatogenesis in mammals. DNA methylation is catalysed by DNA methyltransferase (DNMT) enzymes, which are responsible for the addition of a methyl group to the fifth carbon atom of the cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites. Structurally and functionally five different DNMT enzymes have been identified in mammals, including DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. These enzymes mainly play roles in two DNA methylation processes: maintenance and de novo. While DNMT1 is primarily responsible for maintenance methylation via transferring methyl groups to the hemi-methylated DNA strands following DNA replication, both DNMT3A and DNMT3B are capable of methylating unmodified cytosine residues, known as de novo methylation. However, DNMT3L indirectly participates in de novo methylation, and DNMT2 carries out methylation of the cytosine 38 in the anticodon loop of aspartic acid transfer RNA. To date, many studies have been performed to determine spatial and temporal expression levels and functional features of the DNMT in the male germ cells. This review article comprehensively discusses dynamic expression of the DNMT during spermatogenesis and their relationship with male infertility development in the light of existing investigations.

// Roberto Tirado-Magallanes 1, 2 , Khadija Rebbani 1 , Ricky Lim 1 , Sriharsa Pradhan 3 , Touati Benoukraf 1 1 Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore, Singapore 2 Computational Systems Biology Team, Institut de Biologie de l’Ecole Normale Superieure (IBENS), INSERM, Ecole Normale Superieure, PSL Research University, 75005 Paris, France 3 New England Biolab Inc., Ipswich, MA 01938, USA Correspondence to: Touati Benoukraf, email: benoukraf@nus.edu.sg Keywords: DNA methylation, bisulfite sequencing, gene regulation, chromatin modeling, imprinting Received: August 11, 2016 Accepted: November 07, 2016 Published: November 24, 2016 ABSTRACT The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology.

Deoxyribonucleic acid (DNA) methylation is an epigenetic mechanism involved in the regulation of gene expression and preservation of genome stability in normal cells. Alterations of DNA methylation code have been associated with the development and progression of many diseases, including cancer. Novel technologies allow genome-wide interrogation of methylation patterns. Data on different types of cancer are accumulating; however, elucidation of the consequences of all observed demethylation or methylation events and understanding their associations with other genomic aberrations present great challenges for the future research. Nevertheless, several findings regarding gene silencing by hypermethylation of CpG islands in promoters of genes have been successfully transferred in clinical setting aiding in diagnosis or prognosis of different types of cancer. In addition, therapeutic application of small molecule inhibitors of DNA methyltransferases (DNMTs) to reverse epigenetic silencing has been proven to be beneficial for treatment of various cancers. Key Concepts Alterations of epigenetic mechanisms, including DNA methylation, are implicated in the pathogenesis of many diseases, including cancer. DNA methylation of CpG dinucleotides distributed in the genome can either repress or enable transcription of genes, depending on the location of CpGs and the level of methylation. DNA methylation can be influenced by different internal and external factors, which contribute to the establishment of heterogeneous DNA methylation patterns in cancers. Observed heterogeneity of methylation profiles between tumours and within tumours represents an obstacle in obtaining relevant biological information that could be translated into the knowledge of molecular mechanisms leading to cancer. Advancements in genome-wide methodologies and bioinformatic tools used for deciphering the consequences of altered DNA methylation patterns will enable deeper understanding of molecular mechanisms leading to the development of cancer. Keywords: cancer; CpG island; CIMP phenotype; DNA methylation; epigenetics; gene expression; gene silencing; genomic instability; tumour heterogeneity

SummaryWe analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.

DNA methylation is essential for normal mammalian development and plays critical roles in various biological processes, including genomic imprinting, X-chromosome inactivation and repression of transposable elements. Although DNA methylation patterns are relatively stable in somatic cells, global reprogramming of DNA methylation occurs during mammalian preimplantation development. Advances in DNA methylation profiling techniques have been revealing the DNA methylation dynamics in mammalian embryos. Recently, we and other groups reported genome-scale DNA methylation analyses of human oocytes and preimplantation embryos, highlighting both the similarities and differences in the DNA methylation dynamics between humans and mice. In this review, we introduce the current knowledge of DNA methylation dynamics during early mammalian development. We also discuss the possibility of the application of genome-scale DNA methylation analysis techniques to human gametes and embryos for diagnostic purposes.

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes.

We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant.

SummaryDuring the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.

The DNA methyltransferase enzymes (DNMTs) catalyzing cytosine methylation do so at specific locations of the genome, although with some level of redundancy. The de novo methyltransferases DNMT3A and 3B play a vital role in methylating the genome of the developing embryo in regions devoid of methylation marks. The ability of DNMTs to colocalize at sites of DNA damage is suggestive that recognition of mispaired bases and unusual structures is inherent to the function of these proteins. We provide evidence for G-quadruplex formation within imprinted gene promoters, and report high-affinity binding of recombinant human DNMTs to such DNA G-quadruplexes in vitro. These observations suggest a potential interaction of G-quadruplexes with the DNA methylation machinery, which may be of epigenetic and biological significance.

Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease.

With recent advances in cellular biology, we now appreciate that modifications to DNA and histones can have a profound impact on transcription and function, even in the absence of changes to DNA sequence. These modifications, now commonly referred to as “epigenetic” alterations, have changed how we understand cell behavior, reprogramming and differentiation and have provided significant insight into the mechanisms underlying carcinogenesis. Epigenetic alterations, to this point, are largely identified by changes in DNA methylation and hydroxymethylation as well as methylation, acetylation, and phosphorylation of histone tails. These modifications enable significant flexibility in gene expression, rather than just turning genes “ON” or “OFF.” Herein we describe the epigenetic landscape in the regulation of gene expression with a particular focus on interrogating DNA methylation in myeloid malignancy.

DNA methylation is an important epigenetic modification involved in chromatin organization and gene expression. The function of DNA methylation depends on cell context and is correlated with histone modification patterns. In particular, trimethylation of Lys36 on histone H3 tail (H3K36me3) is associated with DNA methylation and elongation phase of transcription. PWWP domains of the de novo DNA methyltransferases DNMT3A and DNMT3B read this epigenetic mark to guide DNA methylation. Here we report the first crystal structure of the DNMT3B PWWP domain–H3K36me3 complex. Based on this structure, we propose a model of the DNMT3A PWWP domain–H3K36me3 complex and build a model of DNMT3A (PWWP-ADD-CD) in a nucleosomal context. The trimethylated side chain of Lys36 (H3K36me3) is inserted into an aromatic cage similar to the “Royal” superfamily domains known to bind methylated histones. A key interaction between trimethylated Lys36 and a conserved water molecule stabilized by Ser270 explains the lack of affinity of mutated DNMT3B (S270P) for the H3K36me3 epigenetic mark in the ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome. The model of the DNMT3A-DNMT3L heterotetramer in complex with a dinucleosome highlights the mechanism for recognition of nucleosome by DNMT3s and explains the periodicity of de novo DNA methylation.

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces the expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

International audience; Plant DNA methylation that occurs at CG, CHG, and CHH sites (H = A, C, or T) is a hallmark of the repression of repetitive sequences and transposable elements (TEs). The rice (Oryza sativa) genome contains about 40% repetitive sequence and TEs and displays specific patterns of genome-wide DNA methylation. The mechanism responsible for the specific methylation patterns is unclear. Here, we analyzed the function of OsDDM1 (Deficient in DNA Methylation 1) and OsDRM2 (Deficient in DNA Methylation 1) in genome-wide DNA methylation, TE repression, small RNA accumulation, and gene expression. We show that OsDDM1 is essential for high levels of methylation at CHG and, to a lesser extent, CG sites in heterochromatic regions and also is required for CHH methylation that mainly locates in the genic regions of the genome. In addition to a large member of TEs, loss of OsDDM1 leads to hypomethylation and up-regulation of many protein-coding genes, producing very severe growth phenotypes at the initial generation. Importantly, we show that OsDRM2 mutation results in a nearly complete loss of CHH methylation and derepression of mainly small TE-associated genes and that OsDDM1 is involved in facilitating OsDRM2-mediated CHH methylation. Thus, the function of OsDDM1 and OsDRM2 defines distinct DNA methylation pathways in the bulk of DNA methylation of the genome, which is possibly related to the dispersed heterochromatin across chromosomes in rice and suggests that DNA methylation mechanisms may vary among different plant species.

Parental genomes in the endosperm are marked by differential DNA methylation and are therefore epigenetically distinct. This epigenetic asymmetry is established in the gametes and maintained after fertilization by unknown mechanisms. In this manuscript, we have addressed the key question whether parentally inherited differential DNA methylation affects de novo targeting of chromatin modifiers in the early endosperm. Our data reveal that polycomb‐mediated H3 lysine 27 trimethylation (H3K27me3) is preferentially localized to regions that are targeted by the DNA glycosylase DEMETER (DME), mechanistically linking DNA hypomethylation to imprinted gene expression. Our data furthermore suggest an absence of de novo DNA methylation in the early endosperm, providing an explanation how DME‐mediated hypomethylation of the maternal genome is maintained after fertilization. Lastly, we show that paternal‐specific H3K27me3‐marked regions are located at pericentromeric regions, suggesting that H3K27me3 and DNA methylation are not necessarily exclusive marks at pericentromeric regions in the endosperm.

Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal.