Analysis of target sequences of DDM1s in Brassica rapa by MSAP

DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.     

ddm1 plants are sensitive to methyl methane sulfonate and NaCl stresses and are deficient in DNA repair

Plant response to stress includes changes in gene expression and chromatin structure. Our previous work showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational response to stress that included an increase in recombination frequency, cytosine methylation and stress tolerance. It can be hypothesized that changes in chromatin structure are important for an efficient stress response. To test this hypothesis, we analyzed the stress response of ddm1, a mutant impaired in DDM1, a member of the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes. We exposed Arabidopsis thaliana ddm1 mutants to methyl methane sulfonate (MMS) and NaCl and found that these plants were more sensitive. At the same time, ddm1 plants were similar to wild-type plants in sensitivity to temperature and bleomycin stresses. Direct comparison to met1 plants, deficient in maintenance methyltransferase MET1, showed higher sensitivity of ddm1 plants to NaCl. The level of DNA strand breaks upon exposure to MMS increased in wild-type plants but decreased in ddm1 plants. DNA methylation analysis showed that heterozygous ddm1/DDM1 plants had lower methylation as compared to fourth generation of homozygous ddm1/ddm1 plants. Exposure to MMS resulted in a decrease in methylation in wild-type plants and an increase in ddm1 plants. Finally, in vitro DNA excision repair assay showed lower capacity for ddm1 mutant. Our results provided a new example of a link between genetic genome stability and epigenetic genome stability. Key message We demonstrate that heterozygous ddm1/DDM1 plants are more sensitive to stress and have more severe changes in methylation than homozygous ddm1/ddm1 plants.     

Reviewing the Role of DNA (Cytosine-5) Methyltransferase Overexpression in the Cortical GABAergic Dysfunction Associated with Psychosis Vulnerability

In this review, we discuss changes in the regulation of gene expression in the central nervous system (CNS) associated with DNA (cytosine-5) methylation, chromatin remodeling and post-translational covalent modifications of histones.

During brain development, abnormal intrinsic or extrinsic cues may compromise epigenetic processes regulating neural stem cell proliferation and differentiation and thus directly or indirectly could contribute to altered epiphenotypes leading to psychiatric disorders. These mechanisms, that include chromatin remodeling and reversible changes in promoter methylation patterns, are largely expressed by terminally differentiated cortical GABAergic neurons. These neurons are unique among various brain cell subtypes because they express high levels of DNA-methyltransferase-1 (DNMT1). Moreover, DNMT1 expression is further increased in schizophrenia (SZ) and bipolar (BP) disorder brains.

To unravel how this pathological DNMT1 overexpression induces GABAergic neuronal dysfunction in SZ and in other psychoses, we report on how alterations in methylation modify the expression of susceptible vulnerability genes such as reelin or GAD67 in these neurons. The results encourage the view that promoter hypermethylation in GABAergic neurons that occurs in SZ represents a testable target for novel therapeutic strategies to treat this disorder.     

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.     

Effects of exogenous nitric oxide on antioxidation and DNA methylation of <Emphasis Type="Italic">Dendrobium huoshanense</Emphasis> grown under drought stress

Nitric oxide (NO) is an important biological messenger in plants, which has been implicated in response to abiotic stress. To study the effects of exogenous NO on drought menace, the tube seedlings of Dendrobium huoshanense were selected and treated with 10% polyethylene glycol (PEG-6000) to simulate drought stress. After application of sodium nitroprusside (SNP), the relative water content (RWC) and antioxidant enzyme activities were determined. As a result, plant treated with 50 μmol L⁻¹ of SNP maintained high level of RWC and lower content of malondialdehyde (MDA). Furthermore, the antioxidant enzyme activities were obviously enhanced. However, the higher concentration of SNP (100 μmol L⁻¹) enhanced the effects of drought stress for plant. For further analysis of the response mechanism to exogenous NO, the methylation-sensitive amplified polymorphism (MSAP) technique was used to investigate the changes of DNA methylation. When the seedlings of Dendrobium huoshanense were treated with 50 μmol L⁻¹ SNP containing 10% PEG-6000, levels of global DNA methylation of Dendrobium huoshanense were decreased. Nevertheless, the demethylation rate of methylated sites increased, accounting for 12.5% of total methylation sites. These results implied that some expressed genes were involved in the response process to drought stress triggered by NO in Dendrobium huoshanense.     

Inhibition of DNA demethylation enhances plant tolerance to cadmium toxicity by improving iron nutrition

Although the alteration of DNA methylation due to abiotic stresses, such as exposure to the toxic metal cadmium (Cd), has been often observed in plants, little is known about whether such epigenetic changes are linked to the ability of plants to adapt to stress. Herein, we report a close linkage between DNA methylation and the adaptational responses in Arabidopsis plants under Cd stress. Exposure to Cd significantly inhibited the expression of three DNA demethylase genes ROS1/DML2/DML3 (RDD) and elevated DNA methylation at the genome-wide level in Col-0 roots. Furthermore, the profile of DNA methylation in Cd-exposed Col-0 roots was similar to that in the roots of rdd triple mutants, which lack RDD, indicating that Cd-induced DNA methylation is associated with the inhibition of RDD. Interestingly, the elevation in DNA methylation in rdd conferred a higher tolerance against Cd stress and improved cellular Fe nutrition in the root tissues. In addition, lowering the Fe supply abolished improved Cd tolerance due to the lack of RDD in rdd. Together, these data suggest that the inhibition of RDD-mediated DNA demethylation in the roots by Cd would in turn enhance plant tolerance to Cd stress by improving Fe nutrition through a feedback mechanism.     

Stress and aberrant phenotypes in vitro culture

Stress responses are largely conserved in eukaryotic cells, but with plants having certain distinctive reactions to specific stresses, e.g. the induction of pathogenesis-related proteins. General responses to stress involve signaling stress detection via the redox system, checkpoints arresting the cell cycle and DNA repair processes stimulated in response to DNA damage. Specific responses to stress include the induction of protective metabolites, such as betaines, and protective proteins, for example, heat shock proteins. Chemical signals, e.g. reactive oxygen species, Ca²⁺ and plant hormones, acting through signal transduction cascades activate genomic re-programming. Genome plasticity in plants allows adaptation to environmental conditions and includes genomic or epigenetic changes (histone acetylation, methylation, chromatin remodeling etc.) and possibly directed mutation. In plants, recent research has indicated that intricate stress response mechanisms and `cross talk' between stress responses exist. Here, changes in the plant genome and in genomic expression in development and as a response to environmental stress are reviewed as background to a discussion of the basis of aberrant genomic expression in vitro. Markers are discussed which may be used to characterize the stress exposure of in vitro tissues.     

RNAi-independent de novo DNA methylation revealed in Arabidopsis mutants of chromatin remodeling gene DDM1

Methylation of histone H3 lysine 9 (H3K9me) and small RNAs are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non‐CpG sites. Transposon‐specific non‐CpG methylation in plants is controlled by small RNAs and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly demonstrated in plants. We have previously shown that a mutation in the chromatin remodeling gene DDM1 (DECREASE IN DNA METHYLATION 1) induces a global decrease but a local increase of cytosine methylation and accumulation of small RNA at a locus called BONSAI. Here we show that de novo BONSAI methylation does not depend on RNAi but does depend on H3K9me. In mutants of H3K9 methyltransferase gene KRYPTONITE or the H3K9me‐dependent DNA methyltransferase gene CHROMOMETHYALSE3, the ddm1‐induced de novo cytosine methylation was abolished for all three contexts (CpG, CpHpG and CpHpH). Furthermore, RNAi mutants showed strong developmental defects when combined with the ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications that may be conserved among diverse eukaryotes.     

Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification

Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.     

Cumulative lifetime maternal stress and epigenome-wide placental DNA methylation in the PRISM cohort

Evolving evidence links maternal stress exposure to changes in placental DNA methylation of specific genes regulating placental function that may have implications for the programming of a host of chronic disorders. Few studies have implemented an epigenome-wide approach. Using the Infinium HumanMethylation450 BeadChip (450K), we investigated epigenome-wide placental DNA methylation in relation to maternal experiences of traumatic and non-traumatic stressors over her lifetime assessed using the Life Stressor Checklist-Revised (LSC-R) survey (n = 207). We found differential DNA methylation at epigenome-wide statistical significance (FDR = 0.05) for 112 CpGs. Additionally, we observed three clusters that exhibited differential methylation in response to high maternal lifetime stress. Enrichment analyses, conducted at an FDR = 0.20, revealed lysine degradation to be the most significant pathway associated with maternal lifetimes stress exposure. Targeted enrichment analyses of the three largest clusters of probes, identified using the gap statistic, were enriched for genes associated with endocytosis (i.e., SMAP1, ANKFY1), tight junctions (i.e., EPB41L4B), and metabolic pathways (i.e., INPP5E, EEF1B2). These pathways, also identified in the top 10 KEGG pathways associated with maternal lifetime stress exposure, play important roles in multiple physiological functions necessary for proper fetal development. Further, two genes were identified to exhibit multiple probes associated with maternal lifetime stress (i.e., ANKFY1, TM6SF1). The methylation status of the probes belonging to each cluster and/or genes exhibiting multiple hits, may play a role in the pathogenesis of adverse health outcomes in children born to mothers with increased lifetime stress exposure.     

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis

Background

DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear.

Results

We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection.

Conclusions

Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0458-3) contains supplementary material, which is available to authorized users.     

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, the molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in a gain of DNMT1 function. However, a substantial body of data suggested that RFTS is required for DNMT1 activity. Here, we demonstrate that deletion of RFTS alters DNMT1-dependent DNA methylation during malignant transformation. Compared to full-length DNMT1, ectopic expression of hyperactive DNMT1-ΔRFTS caused greater malignant transformation and enhanced promoter methylation with condensed chromatin structure that silenced DAPK and DUOX1 expression. Simultaneously, deletion of RFTS impaired DNMT1 chromatin association with pericentromeric Satellite 2 (SAT2) repeat sequences and produced DNA demethylation at SAT2 repeats and globally. To our knowledge, RFTS-deleted DNMT1 is the first single factor that can reprogram focal hypermethylation and global hypomethylation in parallel during malignant transformation. Our evidence suggests that the RFTS domain of DNMT1 is a target responsible for epigenetic changes in cancer.     

Epigenetic chromatin modifiers in barley: IV. The study of barley Polycomb group (PcG) genes during seed development and in response to external ABA

Background  

Epigenetic phenomena have been associated with the regulation of active and silent chromatin states achieved by modifications of chromatin structure through DNA methylation, and histone post-translational modifications. The latter is accomplished, in part, through the action of PcG (Polycomb group) protein complexes which methylate nucleosomal histone tails at specific sites, ultimately leading to chromatin compaction and gene silencing. Different PcG complex variants operating during different developmental stages have been described in plants. In particular, the so-called FIE/MEA/FIS2 complex governs the expression of genes important in embryo and endosperm development in Arabidopsis. In our effort to understand the epigenetic mechanisms regulating seed development in barley (Hordeum vulgare), an agronomically important monocot plant cultivated for its endosperm, we set out to characterize the genes encoding barley PcG proteins.