Evaluation of the Properties of the DNA Methyltransferase from Aeropyrum pernix K1.

Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.

DNA methyltransferase CHROMOMETHYLASE3 prevents ONSEN transposon silencing under heat stress.

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

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

Ectopic targeting of CG DNA methylation in Arabidopsis with the bacterial SssI methyltransferase.

The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.

In Arabidopsis thaliana Cd differentially impacts on hormone genetic pathways in the methylation defective ddc mutant compared to wild type.

DNA methylation plays an important role in modulating plant growth plasticity in response to stress, but mechanisms involved in such control need further investigation. We used drm1 drm2 cmt3 mutant of Arabidopsis thaliana, defective in DNA methylation, to explore metabolic pathways downstream epigenetic modulation under cadmium (Cd) stress. To this aim, a transcriptomic analysis was performed on ddc and WT plants exposed to a long-lasting (21 d) Cd treatment (25/50 µM), focusing on hormone genetic pathways. Growth parameters and hormones amount were also estimated. Transcriptomic data and hormone quantification showed that, under prolonged Cd treatment, level and signalling of growth-sustaining hormones (auxins, CKs, GAs) were enhanced and/or maintained, while a decrease was detected for stress-related hormones (JA, ABA, SA), likely as a strategy to avoid the side effects of their long-lasting activation. Such picture was more effective in ddc than WT, already at 25 µM Cd, in line with its better growth performance. A tight relationship between methylation status and the modulation of hormone genetic pathways under Cd stress was assessed. We propose that the higher genome plasticity conferred to ddc by DNA hypomethylated status underlies its prompt response to modulate hormones genetic pathways and activity and assure a flexible growth.

Genome-wide identification and analysis of DNA methyltransferase and demethylase gene families in Dendrobium officinale reveal their potential functions in polysaccharide accumulation.

BACKGROUND: DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale. RESULTS: In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased. CONCLUSIONS: These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.

DNA Geminivirus Infection Induces an Imprinted E3 Ligase Gene to Epigenetically Activate Viral Gene Transcription.

Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

Natural variation in DNA methylation homeostasis and the emergence of epialleles.

In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns in Arabidopsis thaliana are highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725 A. thaliana accessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CG methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis.

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

Age-related Changes in the Global DNA Methylation Profile of Oligodendrocyte Progenitor Cells Derived from Rat Spinal Cords.

Demyelination of axons plays an important role in the pathology of many spinal cord diseases and injuries. Remyelination in demyelinated lesions is primarily performed by oligodendrocyte progenitor cells (OPCs), which generate oligodendrocytes in the developing and mature central nervous system. The efficiency of remyelination decreases with age. Many reports suggest that this decline in remyelination results from impaired OPC recruitment and differentiation during aging. Of the various molecular mechanisms involved in aging, changes in epigenetic modifications have received particular attention. Global DNA methylation is a major epigenetic modification that plays important roles in cellular senescence and organismal aging. Thus, we aimed to evaluate the dynamic changes in the global DNA methylation profiles of OPCs derived from rat spinal cords during the aging process. We separated and cultured OPCs from the spinal cords of neonatal, 4-month-old, and 16-month-old rats and investigated the age-related alterations of genomic DNA methylation levels by using quantitative colorimetric analysis. To determine the potential cause of dynamic changes in global DNA methylation, we further analyzed the activity of DNA methyltransferases (DNMTs) and the expression of DNMT1, DNMT3a, DNMT3b, TET1, TET2, TET3, MBD2, and MeCP2 in the OPCs from each group. Our results showed the genomic DNA methylation level and the activity of DNMTs from OPCs derived from rat spinal cords decreased gradually during aging, and OPCs from 16-month-old rats were characterized by global hypomethylation. During OPC aging, the mRNA and protein expression levels of DNMT3a, DNMT3b, and MeCP2 were significantly elevated; those of DNMT1 were significantly down-regulated; and no significant changes were observed in those for TET1, TET2, TET3, or MBD2. Our results indicated that global DNA hypomethylation in aged OPCs is correlated with DNMT1 downregulation. Together, these data provide important evidence for partly elucidating the mechanism of age-related impaired OPC recruitment and differentiation and assist in the development of new treatments for promoting efficient remyelination.

Effects of mine tailings exposure on early life stages of atlantic cod.

In Norway, mine tailings waste can be deposited by coastal submarine dispersal. Mine tailings slurry includes fine particles <10 µm with elevated levels of metals (e.g., copper, iron) from residual mineral ore. Prolonged suspension of small particles in the water column may bring them into contact with locally spawned pelagic fish eggs, including Atlantic cod, Gadus morhua. Newly fertilized cod embryos were exposed to suspended mine tailings particles up to 3.2 mg/L in flow-through aquaria for a total of 21 d. Significantly more particles adhered to the surface of the chorion from the high treatment after 11-d exposure, and dissolved Cu concentrations increased in the water (up to 0.36 ± 0.06 µg/L). There was no adverse effect on embryo mortality but an 8% elevation in larval mortality. There were no differences with treatment on timing of hatching, embryo and larva morphometrics, abnormalities, or cardiac activity. There was a treatment-dependent up-regulation of stress marker genes (hspa8, cyp1c1) but no indication of metal-induced activation of metallothionien (mt gene transcription). Transcription markers for DNA and histone methyltransferases did show treatment-related up-regulation, indicative of altered methylation in larvae when developmental methylation patterns are determined, indicating some level of chronic toxicity that may have longer-term effects. Environ Toxicol Chem 2019;38:1446-1454. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Hypomethylated drm1 drm2 cmt3 mutant phenotype of Arabidopsis thaliana is related to auxin pathway impairment.

DNA methylation carried out by different methyltransferase classes is a relevant epigenetic modification of DNA which plays a relevant role in the development of eukaryotic organisms. Accordingly, in Arabidopsis thaliana loss of DNA methylation due to combined mutations in genes encoding for DNA methyltransferases causes several developmental abnormalities. The present study describes novel growth disorders in the drm1 drm2 cmt3 triple mutant of Arabidopsis thaliana, defective both in maintenance and de novo DNA methylation, and highlights the correlation between DNA methylation and the auxin hormone pathway. By using an auxin responsive reporter gene, we discovered that auxin accumulation and distribution were affected in the mutant compared to the wild type, from embryo to adult plant stage. In addition, we demonstrated that the defective methylation status also affected the expression of genes that regulate auxin hormone pathways from synthesis to transport and signalling and a direct relationship between differentially expressed auxin-related genes and altered auxin accumulation and distribution in embryo, leaf and root was observed. Finally, we provided evidence of the direct and organ-specific modulation of auxin-related genes through the DNA methylation process.

Glucocorticoid Receptor Stimulation Resulting from Early Life Stress Affects Expression of DNA Methyltransferases in Rat Prefrontal Cortex.

Early life stress initiates long-term neurobiological changes that affect stress resilience and increased susceptibility to psychopathology. Maternal separation (MS) is used to cause early life stress and it induces profound neurochemical and behavioral changes that last until adulthood. The molecular pathways of how MS affects the regulation of DNA methyltransferases (Dnmt) in brain have not been entirely characterized. We evaluated MS effects on Dnmt1, Dnmt3a and Dnmt3b expression, DNMT enzyme activity and glucocorticoid receptor (GR) recruitment to different Dnmt loci in the prefrontal cortex (PFC) of Wistar rats. We found increased plasma corticosterone levels after MS that were associated with induced Dnmt expression and enzyme activity in rat PFC at post-natal day 15 (PND15). Chromatin immunoprecipitation showed increased binding of GR at the Dnmt3b promoter after MS, suggesting that genomic signaling of GR is an important regulatory mechanism for the induced Dnmt3b expression and DNMT activity. Although GR also binds to Dnmt3a promoter and a putative regulatory region in intron 3 in rat PFC, its expression after maternal separation may be influenced by other mechanisms. Therefore, GR could be a link between early life stress experience and long-term gene expression changes induced by aberrant DNA methylation.

Association between the estrogen receptor α gene polymorphisms rs2234693 and rs9340799 and severe and mild pre-eclampsia: a meta-analysis.

This meta-analysis was performed in order to determine the associations between the estrogen receptor α (ESR1) gene PvuII site (-397T/C, rs2234693) and XbaI site (-351A/G, rs9340799) polymorphisms with severe and mild pre-eclampsia. Eligible studies were identified by searching PubMed, Medline, Embase, China National Knowledge Infrastructure (CNKI), and WanFang databases until May 2018. The pooled odds ratio (OR) and 95% confidence interval (CI) were used to calculate the associations. Six articles (consisting of seven studies; one article was considered as two separate studies with two different subpopulations) investigated the ESR1 gene PvuII -397T/C and XbaI -351A/G polymorphisms in severe and mild pre-eclampsia patients and included controls. The pooled results indicated an increased risk of severe pre-eclampsia for the XbaI -351A/G polymorphism (OR = 1.67, 95% CI = 1.10-2.25, P=0.017 for GG compared with AA+GA; OR = 1.81, 95% CI = 1.17-2.82, P=0.008 for GG compared with GA). The GG genotype of the ESR1 XbaI polymorphism could be a genetic risk factor for severe pre-eclampsia susceptibility. However, the ESR1 gene PvuII -397T/C polymorphism was not significantly associated with the risk of severe pre-eclampsia, and there was no association between mild pre-eclampsia and the ESR1 gene PvuII -397T/C and XbaI -351A/G polymorphisms separately. The current meta-analysis indicates that the ESR1 XbaI genetic polymorphism may be associated with severe pre-eclampsia. However, there was no association of the ESR1 gene PvuII and XbaI polymorphisms with the risk of mild pre-eclampsia. Owing to the low statistical power, the results may not be sufficiently robust and this conclusion should be interpreted cautiously, which highlights the requirement for large-scale and high-quality studies in this field.

CMT3 and SUVH4/KYP silence the exonic Evelknievel retroelement to allow for reconstitution of CMT1 mRNA.

BACKGROUND: The Chromomethylase 1 (CMT1) has long been considered a nonessential gene because, in certain Arabidopsis ecotypes, the CMT1 gene is disrupted by the Evelknievel (EK) retroelement, inserted within exon 13, or contains frameshift mutations, resulting in a truncated, non-functional protein. In contrast to other transposable elements, no transcriptional activation of EK was observed under stress conditions (e.g., protoplasting). RESULTS: We wanted to explore the regulatory pathway responsible for EK silencing in the Ler ecotype and its effect on CMT1 transcription. Methylome databases confirmed that EK retroelement is heavily methylated and methylation is extended toward CMT1 downstream region. Strong transcriptional activation of EK accompanied by significant reduction in non-CG methylation was found in cmt3 and kyp2, but not in ddm1 or RdDM mutants. EK activation in cmt3 and kyp2 did not interfere with upstream CMT1 expression but abolish transcription through the EK. We identified, in wild-type Ler, three spliced variants in which the entire EK is spliced out; one variant (25% of splicing incidents) facilitates proper reconstitution of an intact CMT1 mRNA. We could recover very low amount of the full-length CMT1 mRNA from WT Ler and Col, but not from cmt3 mutant. CONCLUSIONS: Our findings highlight CMT3-SUVH4/KYP as the major pathway silencing the intragenic EK via inducing non-CG methylation. Furthermore, retroelement insertion within exons (e.g., CMT1) may not lead to a complete abolishment of the gene product when the element is kept silent. Rather the element can be spliced out to bring about reconstruction of an intact, functional mRNA and possibly retrieval of an active protein.

Association Between Schizophrenia and DNA Demethylase Activity in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: DNA demethylase is a crucial enzyme in the epigenetic modification and regulation mechanisms of gene transcription. Based on previous assertions that the pathophysiology of schizophrenia is associated with epigenetics, we aimed to explore whether DNA demethylase activity might be related to schizophrenia in northeast China. METHODS: We recruited 25 patients with first-episode schizophrenia and 29 normal controls from a northeast Chinese Han population. The diagnostic criteria of schizophrenia were determined according to diseases and related health problems, the tenth revision (ICD-10), and criteria of mental disorders, the third revised edition (CCMD3). DNA demethylase activity in human peripheral blood mononuclear cells (PBMCs) was measured using a DNA demethylase activity colorimetric assay ultra kit. RESULTS: Using Student's t-test, activation of DNA demethylase and its activity were higher in schizophrenia patients compared to healthy individuals (p < 0.001). Furthermore, the level of DNA demethylase activity in male and female subjects with schizophrenia significantly increased (all p < 0.05). CONCLUSIONS: Our data showed that DNA demethylase might play a role in the pathophysiology of schizophrenia, and individuals with higher DNA demethylase activity were susceptible to schizophrenia in a northeast Chinese Han population. To the best of our knowledge, this is the first time directly measured human blood samples to examine the association between first-episode schizophrenia patients and DNA demethylase activity, which will provide new insight to explore the effect on the mechanism of schizophrenia.

Atrazine exposure decreases the activity of DNMTs, global DNA methylation levels, and dnmt expression.

Atrazine, a herbicide used on agricultural crops is widely applied in the Midwestern United States as well as other areas of the globe. Atrazine frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. Previous studies have reported morphological, hormonal, and molecular alterations due to developmental and adulthood atrazine exposure; however, studies examining epigenetic alterations are limited. In this study, the effects of atrazine exposure on DNA methyltransferase (DNMT) activity and kinetics were evaluated. Global DNA methylation levels and dnmt expression in zebrafish larvae exposed to 0, 3, or 30 parts per billion (ppb) atrazine throughout embryogenesis was then assessed. Results indicate that atrazine significantly decreased the activity of maintenance DNMTs and that the inhibition mechanism can be described using non-competitive Michaelis-Menten kinetics. Furthermore, results show that an embryonic atrazine exposure decreases global methylation levels and the expression of dnmt4 and dnmt5. These findings indicate that atrazine exposure can decrease the expression and activity of DNMTs, leading to decreased DNA methylation levels.

Characterization of Vsr endonucleases from Neisseria meningitidis.

DNA methylation is a common modification occurring in all living organisms. 5-methylcytosine, which is produced in a reaction catalysed by C5-methyltransferases, can spontaneously undergo deamination to thymine, leading to the formation of T:G mismatches and C→T transitions. In Escherichia coli K-12, such mismatches are corrected by the Very Short Patch (VSP) repair system, with Vsr endonuclease as the key enzyme. Neisseria meningitidis possesses genes that encode DNA methyltransferases, including C5-methyltransferases. We report on the mutagenic potential of the meningococcal C5-methyltransferases M.NmeDI and M.NmeAI resulting from deamination of 5-methylcytosine. N. meningitidis strains also possess genes encoding potential Vsr endonucleases. Phylogenetic analysis of meningococcal Vsr endonucleases indicates that they belong to two phylogenetically distinct groups (type I or type II Vsr endonucleases). N. meningitidis serogroup C (FAM18) is a representative of meningococcal strains that carry two Vsr endonuclease genes (V.Nme18IIP and V.Nme18VIP). The V.Nme18VIP (type II) endonuclease cut DNA containing T:G mismatches in all tested nucleotide contexts. V.Nme18IIP (type I) is not active in vitro, but the change of Tyr69 to His69 in the amino acid sequence of the protein restores its endonucleolytic activity. The presence of tyrosine in position 69 is a characteristic feature of type I meningococcal Vsr proteins, while type II Vsr endonucleases possess His69. In addition to the T:G mismatches, V.Nme18VIP and V.Nme18IIPY69H recognize and digest DNA with T:T or U:G mispairs. Thus, for the first time, we demonstrate that the VSP repair system may have a wider significance and broader substrate specificity than DNA lesions that only result from 5-methylcytosine deamination.