DNA Methylase Research Articles

From sequence to significance: A thorough investigation of the distinctive genome features uncovered in C. Werkmanii strain NIB003

Citrobacter werkmanii (C. werkmanii), an opportunistic urinary bacterium that causes diarrhea, is poorly understood. Our research focuses on genetic features that are crucial to disease development, such as pathogenic interactions, antibiotic resistance, virulence genes and genetic variation. Following its morphological, biochemical, and molecular identification, the whole genome of C. werkmanii strain NIB003 was sequenced in Bangladesh for the first time. Despite having around 80% whole genome conservation, the research shows that the Bangladeshi strain forms a separate phylogenetic cluster. This emphasises the genetic variability within C. werkmanii, resulting in particular modifications at the strain level and changes in its ability to cause disease. The results of the genetic diversity analysis indicate that the Bangladeshi sequenced genome is more diverse than the other strains due to the existence of unique features, such as the presence of t-RNA binding domain and N-6 adenine-specific DNA methylases.

Unraveling the Role of Bromodomain and Extra-Terminal Proteins in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma, associated with poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is limited. Bromodomain and extra-terminal (BET) proteins are involved in both physiological and pathological events. However, the role of BET proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BET protein family members, BRD2, BRD3, and BRD4, are aberrantly overexpressed in uLMS tissues compared to the myometrium, with a significant change by histochemical scoring assessment. Furthermore, inhibiting BET proteins with their small, potent inhibitors (JQ1 and I-BET 762) significantly inhibited the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-sequencing analysis revealed that the inhibition of BET proteins with JQ1 and I-BET 762 altered several critical pathways, including the hedgehog pathway, EMT, and transcription factor-driven pathways in uLMS. In addition, the targeted inhibition of BET proteins altered several other epigenetic regulators, including DNA methylases, histone modification, and m6A regulators. The connections between BET proteins and crucial biological pathways provide a fundamental structure to better understand uterine diseases, particularly uLMS pathogenesis. Accordingly, targeting the vulnerable epigenome may provide an additional regulatory mechanism for uterine cancer treatment.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

DNA methylase 1 influences temperature responses and development in the invasive pest Tuta absoluta.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absolutaMeyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

Revealing role of epigenetic modifiers and DNA oxidation in cell-autonomous regulation of Cancer stem cells

BackgroundBreast cancer cells (BCCs) can remain undetected for decades in dormancy. These quiescent cells are similar to cancer stem cells (CSCs); hence their ability to initiate tertiary metastasis. Dormancy can be regulated by components of the tissue microenvironment such as bone marrow mesenchymal stem cells (MSCs) that release exosomes to dedifferentiate BCCs into CSCs. The exosomes cargo includes histone 3, lysine 4 (H3K4) methyltransferases - KMT2B and KMT2D. A less studied mechanism of CSC maintenance is the process of cell-autonomous regulation, leading us to examine the roles for KMT2B and KMT2D in sustaining CSCs, and their potential as drug targets.MethodsUse of pharmacological inhibitor of H3K4 (WDR5–0103), knockdown (KD) of KMT2B or KMT2D in BCCs, real time PCR, western blot, response to chemotherapy, RNA-seq, and flow cytometry for circulating markers of CSCs and DNA hydroxylases in BC patients. In vivo studies using a dormancy model studied the effects of KMT2B/D to chemotherapy.ResultsH3K4 methyltransferases sustain cell autonomous regulation of CSCs, impart chemoresistance, maintain cycling quiescence, and reduce migration and proliferation of BCCs. In vivo studies validated KMT2’s role in dormancy and identified these genes as potential drug targets. DNA methylase (DNMT), predicted within a network with KMT2 to regulate CSCs, was determined to sustain circulating CSC-like in the blood of patients.ConclusionH3K4 methyltransferases and DNA methylation mediate cell autonomous regulation to sustain CSC. The findings provide crucial insights into epigenetic regulatory mechanisms underlying BC dormancy with KMT2B and KMT2D as potential therapeutic targets, along with standard care. Stem cell and epigenetic markers in circulating BCCs could monitor treatment response and this could be significant for long BC remission to partly address health disparity.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques.Key points• Non-switchable methylases always inhibit the relevant type IIS endonuclease activity• Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site• Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assemblyGraphical

The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single-base resolution methylation profiles of the Pacific oyster, including female-to-male (FMa-to-FMb) and male-to-female (MFa-to-MFb) sex reversal, as well as sex non-reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during female-to-male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male-to-female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox-like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non-reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.

DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in Osmanthus fragrans.

Short-term ambient low temperature (ALT) stimulation is necessary for Osmanthus fragrans to facilitate continued flower opening after floral bud development reaches maturity. DNA methylation, a vital epigenetic modification, regulates various biological processes in response to temperature fluctuations. However, its role in temperature-driven flower opening remains elusive. In this study, we identified the pivotal timeframe during which O. fragrans promptly detected temperature cues. Using whole-genome bisulfite sequencing, we explored global DNA hypomethylation during this phase, with the most significant changes occurring in CHH sequence contexts. Auxin transport inhibitor (TIBA) application revealed that ALT-induced endogenous auxin accumulation promoted peduncle elongation. In our mRNA-seq analysis, we discovered that the differentially expressed genes (DEGs) with hypo-differentially methylated regions (hypo-DMRs) were mainly enriched in auxin and temperature response, RNA processing, and carbohydrate and lipid metabolism. Transcripts of three DNA demethylase genes (OfROS1a, OfDML3, OfDME) showed upregulation. Furthermore, all DNA methylase genes, except OfCMT2b, also displayed increased expression, specifically with two of them, OfCMT3a and OfCMT1, being associated with hypo-DMRs. Promoter assays showed that OfROS1a, with promoters containing low-temperature- and auxin-responsive elements, were activated by ALT and exogenous IAA at low concentrations but inhibited at high concentrations. Overexpression of OfROS1 reduced endogenous auxin levels but enhanced the expression of genes related to auxin response and spliceosome in petunia. Furthermore, OfROS1 promoted sucrose synthesis in petunia corollas. Our data characterized the rapid response of active DNA hypomethylation to ALT and suggested a possible epiregulation of temperature-dependent flower opening in O. fragrans. This study revealed the pivotal role of DNA hypomethylation in O. fragrans during the ALT-responsive phase before flower opening, involving dynamic DNA demethylation, auxin signaling modulation, and a potential feedback loop between hypomethylation and methylation.

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine.

Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

Dysregulation of DNA epigenetic modulators during prostate carcinogenesis in an eastern Indian patient population: Prognostic implications

The role of epigenetic alteration in prostate cancer pathogenesis was reported. We aimed to analyze dysregulation of DNA methylase (DNA methyl transferase/DNMT) and demethylase (ten eleven translocase/TET) and the associated interplay between them during prostate tumorigenesis. Promoter methylation and RNA/protein expression of selected DNMT and TETs were analysed in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Genomic 5-hydroxymethylcytosine (5hmC) level was detected and correlated with DNMT and TET proteins. Clinicopathological association of molecular data was done. Our data revealed a very low frequency of promoter methylation for DNMT1 (5-3% and high frequency for TET1 (22–38%), TET2 (68–90 %), and TET3 (43-32 %) in BPH and PCa. The promoter methylation of DNMT1 (p = 0.019) showed a significantly decreasing trend, while that of TET1 (p = 0.0005) and TET2 (p < 0.0001) showed an increasing trend from normal prostate to BPH to PCa, indicating their epigenetic dysregulation during prostate tumorigenesis. RNA/protein overexpression of DNMT1 and reduced expression of TET1 and TET2 in PCa compared to BPH were associated with the promoter methylation status of genes. The 5hmC level was significantly lower in PCa than in BPH and correlated negatively with DNMT1 but positively with TET1 and TET2 proteins, suggesting dysregulation of DNA methylase and de-methylase activities during prostate tumorigenesis. Lastly, tumors having methylated TET1 and TET2 promoters showed advanced clinicopathological features (a higher PSA level/Gleason score) and increased risk of bone metastasis. In conclusion, DNMT1 upregulation and epigenetic silencing of TET1 and TET2 was seen during PCa development. TET1 and TET2 promoter methylation has prognostic importance.

Crucial roles of trehalose and 5-azacytidine in alleviating salt stress in tomato: Both synergistically and independently

Trehalose may improve plant stress tolerance by regulating gene expression under different abiotic stresses. DNA methylation is involved in plant growth and development, but also in response to abiotic stresses. 5-azacytidine is a widely used inhibitor of DNA methylation. In this study, tomato (Solanum lycopersicum L. ‘Ailsa Craig’) was used as experimental material to explore the effects of trehalose and DNA methylation on the growth and development in tomato seedlings under salt stress. 10 mM trehalose, 50 μM 5-azacytidine, and their combined treatments could significantly increase growth parameters in tomato under salt stress, indicating trehalose and 5-azacytidine might play crucial roles in alleviating salt stress both synergistically and independently. Additionally, trehalose significantly down-regulated the expression of DNA methylase genes (SlDRM5, SlDRM1L1, SlCMT3 and SlCMT2) and up-regulated the expression of DNA demethylases genes under salt stress, suggesting that trehalose might regulate DNA methylation under salt stress condition. Under salt stress, trehalose and 5-azacytidine treatments enhanced antioxidant enzyme activity and induced antioxidant enzyme gene expression in tomato seedlings. Meanwhile, trehalose and 5-azacytidine increased ABA content by regulating the expression of ABA metabolism-related genes, thereby enhancing salt tolerance in tomato. Altogether, these results suggest that trehalose conferred salt tolerance in tomato seedlings probably by DNA demethylation and enhancing antioxidant capability and ABA accumulation.

Casticin as potential anticancer agent: recent advancements in multi-mechanistic approaches.

Plants, with their range of pharmacologically active molecules, represent the most promising source for the production of new anticancer drugs and for the formulation of adjuvants in chemotherapy treatments to reduce drug content and/or counteract the side effects of chemotherapy. Casticin is a major bioactive flavonoid isolated from several plants, mainly from the Vitex species. This compound is well known for its anti-inflammatory and antioxidant properties, which are mainly exploited in traditional medicine. Recently, the antineoplastic potential of casticin has attracted the attention of the scientific community for its ability to target multiple cancer pathways. The purpose of this review is, therefore, to present and critically analyze the antineoplastic potential of casticin, highlighting the molecular pathways underlying its antitumor effects. Bibliometric data were extracted from the Scopus database using the search strings "casticin" and "cancer" and analyzed using VOSviewer software to generate network maps to visualize the results. Overall, more than 50% of the articles were published since 2018 and even more recent studies have expanded the knowledge of casticin's antitumor activity by adding interesting new mechanisms of action as a topoisomerase IIα inhibitor, DNA methylase 1 inhibitor, and an upregulator of the onco-suppressive miR-338-3p. Casticin counteracts cancer progression through the induction of apoptosis, cell cycle arrest, and metastasis arrest, acting on several pathways that are generally dysregulated in different types of cancer. In addition, they highlight that casticin can be considered as a promising epigenetic drug candidate to target not only cancer cells but also cancer stem-like cells.

CG hypermethylation of the bHLH39 promoter regulates its expression and Fe deficiency responses in tomato roots.

Iron (Fe) is an essential micronutrient for all organisms, including plants, whose limited bioavailability restricts plant growth, yield, and nutritional quality. While the transcriptional regulation of plant responses to Fe deficiency have been extensively studied, the contribution of epigenetic modulations, such as DNA methylation, remains poorly understood. Here, we report that treatment with a DNA methylase inhibitor repressed Fe deficiency-induced responses in tomato (Solanum lycopersicum) roots, suggesting the importance of DNA methylation in regulating Fe deficiency responses. Dynamic changes in the DNA methylome in tomato roots responding to short-term (12hours) and long-term (72hours) Fe deficiency identified many differentially methylated regions (DMRs) and DMR-associated genes. Most DMRs occurred at CHH sites under short-term Fe deficiency, whereas they were predominant at CG sites following long-term Fe deficiency. Furthermore, no correlation was detected between the changes in DNA methylation levels and the changes in transcript levels of the affected genes under either short-term or long-term treatments. Notably, one exception was CG hypermethylation at the bHLH39 promoter, which was positively correlated with its transcriptional induction. In agreement, we detected lower CG methylation at the bHLH39 promoter and lower bHLH39 expression in MET1-RNA interference lines compared with wild-type seedlings. Virus-induced gene silencing of bHLH39 and luciferase reporter assays revealed that bHLH39 is positively involved in the modulation of Fe homeostasis. Altogether, we propose that dynamic epigenetic DNA methylation in the CG context at the bHLH39 promoter is involved in its transcriptional regulation, thus contributing to the Fe deficiency response of tomato.

DNMT1 mediates proliferation, migration and invasion of extravillous trophoblasts by regulating the methylation level of APLNR

IntroductionProliferation, migration and invasion of extravillous trophoblasts (EVTs) play an important role in the progression of preeclampsia (PE). The purpose of this study was to investigate the molecular mechanism by which DNA methylase regulates the transcription level of APLNR and affects the phenotypic function of EVTs. Materials and methodsPE mice model and H/R model in HTR8/Svneo cells were constructed. Clinical samples of normal pregnant women and PE patients were collected. Expression and methylation level of APLNR in vivo and in vitro were detected. ChIP-qPCR was used to detect the binding of DNA methyltransferase at the APLNR promoter. The expression of DNA methyltransferase 1 (DNMT1), NO and eNOS in vitro were detected. EVTs proliferation, migration and invasion in vitro were detected. ResultsIn placental tissues or HTR8/Svneo cells of the PE model group, the expression of APLNR was reduced and APLNR methylation level was up-regulated. There was no significant difference in the APLNR expression in placental tissues between normal pregnant women and PE patients. H/R conditions only promote the binding of DNMT1 at the APLNR promoter. DNMT1 interference decreased the enrichment degree of DNMT1 in APLNR promoter region and up-regulated the mRNA and protein levels of APLNR in vivo and in vitro. The activation of APLNR by Elabela (ELA) can promote eNOS transcription, thereby promoting cell proliferation and NO level, while eNOS inhibitor can reverse this effect. DNMT1 down-regulation inhibted APLNR methylation level, promoted eNOS transcription, and promoted EVTs proliferation, migration and invasion, which could be revised by the interference of APLNR. DiscussionDNMT1 promotes eNOS transcription by inhibting APLNR methylation level, and promotes EVTs proliferation, migration and invasion, thus providing a new and broad application prospect for PE treatment.

Impacts of DNA methylases and demethylases on the methylation and expression of Arabidopsis ethylene signal pathway genes.

Arabidopsis ethylene (ET) signal pathway plays important roles in various aspects. Cytosine DNA methylation is significant in controlling gene expression in plants. Here, we analyzed the bisulfite sequencing and mRNA sequencing data from Arabidopsis (de)methylase mutants met1, cmt3, drm1/2, ddm1, ros1-4, and rdd to investigate how DNA (de)methylases influence the DNA methylation and expression of Arabidopsis ET pathway genes. At least 32 genes are found to involved in Arabidopsis ET pathway by text mining. Among them, 14 genes are unmethylated or methylated with very low levels. ACS6 and ACS9 are conspicuously methylated within their upstream regions. The other 16 genes are predominantly methylated at the CG sites within gene body regions in wild-type plants, and mutation of MET1 resulted in almost entire elimination of the CG methylations. In addition, CG methylations within some genes are jointly maintained by MET1 and other (de)methylases. Analyses of mRNA-seq data indicated that some ET pathway genes were differentially expressed between wild-type and diverse mutants. PDF1.2, the marker gene of ET signal pathway, was found being regulated indirectly by the methylases. Eighty-two transposable elements (TEs) were identified to be associated to 15 ET pathway genes. ACS11 is found located in a heterochromatin region that contains 57 TEs, indicating its specific expression and regulation. Together, our results suggest that DNA (de)methylases are implicated in the regulation of CG methylation within gene body regions and transcriptional activity of some ET pathway genes and that maintenance of normal CG methylation is essential for ET pathway in Arabidopsis.

Effects of DNA methylase inhibitors in a murine model of severe BPD

DNA methylation is necessary for developmental gene regulation, but adverse environments result in aberrant methylation and gene silencing. The current pilot study tested the hypothesis that treatment with DNA methylation inhibitors (decitabine; RG108) would improve alveolarization in a newborn murine model of severe bronchopulmonary dysplasia. Newborn mice exposed to maternal inflammation (LPS) and neonatal hyperoxia (85% O2) were treated with decitabine (p3, 0.1 mg/kg; p2, 4, 6, 0.1 mg/kg; or p2, 4, 6, 0.15 mg/kg) or RG108 (p3, 0.0013 mg/kg) delivered intranasally. Modest improvements in alveolarization were observed with decitabine, but no differences were observed with RG108. Attenuated phospho-SMAD2/3 levels and greater surfactant protein C protein levels compared to vehicle were observed with some tested doses. No detrimental side effects were observed with the doses used in this study. In summary, our pilot investigations identified a safe dose for intranasal administration of both methylation inhibitors and provides a foundation for further studies into methylation inhibitors in the context of neonatal lung injury.

Bacterial DNA methyltransferase: A key to the epigenetic world with lessons learned from proteobacteria.

Epigenetics modulates expression levels of various important genes in both prokaryotes and eukaryotes. These epigenetic traits are heritable without any change in genetic DNA sequences. DNA methylation is a universal mechanism of epigenetic regulation in all kingdoms of life. In bacteria, DNA methylation is the main form of epigenetic regulation and plays important roles in affecting clinically relevant phenotypes, such as virulence, host colonization, sporulation, biofilm formation et al. In this review, we survey bacterial epigenomic studies and focus on the recent developments in the structure, function, and mechanism of several highly conserved bacterial DNA methylases. These methyltransferases are relatively common in bacteria and participate in the regulation of gene expression and chromosomal DNA replication and repair control. Recent advances in sequencing techniques capable of detecting methylation signals have enabled the characterization of genome-wide epigenetic regulation. With their involvement in critical cellular processes, these highly conserved DNA methyltransferases may emerge as promising targets for developing novel epigenetic inhibitors for biomedical applications.

Pangenomic analysis of Wolbachia provides insight into the evolution of host adaptation and cytoplasmic incompatibility factor genes.

The genus Wolbachia provides a typical example of intracellular bacteria that infect the germline of arthropods and filarial nematodes worldwide. Their importance as biological regulators of invertebrates, so it is particularly important to study the evolution, divergence and host adaptation of these bacteria at the genome-wide level. Here, we used publicly available Wolbachia genomes to reconstruct their evolutionary history and explore their adaptation under host selection. Our findings indicate that segmental and single-gene duplications, such as DNA methylase, bZIP transcription factor, heat shock protein 90, in single monophyletic Wolbachia lineages (including supergroups A and B) may be responsible for improving the ability to adapt to a broad host range in arthropod-infecting strains. In contrast to A strains, high genetic diversity and rapidly evolving gene families occur in B strains, which may promote the ability of supergroup B strains to adapt to new hosts and their large-scale spreading. In addition, we hypothesize that there might have been two independent horizontal transfer events of cif genes in two sublineages of supergroup A strains. Interestingly, during the independent evolution of supergroup A and B strains, the rapid evolution of cif genes in supergroup B strains resulted in the loss of their functional domain, reflected in a possible decrease in the proportion of induced cytoplasmic incompatibility (CI) strains. This present study highlights for reconstructing of evolutionary history, addressing host adaptation-related evolution and exploring the origin and divergence of CI genes in each Wolbachia supergroup. Our results thus not only provide a basis for further exploring the evolutionary history of Wolbachia adaptation under host selection but also reveal a new research direction for studying the molecular regulation of Wolbachia- induced cytoplasmic incompatibility.

Comparative analysis of the chrysanthemum transcriptome with DNA methylation inhibitors treatment and silencing MET1 lines

BackgroundAs one of the ten most famous flowers in China, the chrysanthemum has rich germplasm with a variety of flowering induction pathways, most of which are photoperiod-induced. After treatment with DNA methylation inhibitors, it was found that DNA methylation plays an important role in flowering regulation, but the mechanism of action remains unclear. Therefore, in this study, curcumin, 5-azaC, their mixed treatment, and MET1-RNAi lines were used for transcriptome sequencing to find out how different treatments affected gene expression in chrysanthemums at different stages of flowering.ResultsGenomic DNA methylation levels were measured using HPLC technology. The methylation level of the whole genome in the vegetative growth stage was higher than that in the flowering stage. The methylation level of DNA in the vegetative growth stage was the lowest in the curcumin and mixed treatment, and the methylation level of DNA in the transgenic line, mixed treatment, and curcumin treatment was the lowest in the flowering stage. The flowering rate of mixed treatment and curcumin treatment was the lowest. Analysis of differentially expressed genes in transcriptomes showed that 5-azaC treatment had the most differentially expressed genes, followed by curcumin and transgenic lines, and mixed treatment had the fewest. In addition, 5-azaC treatment resulted in the differential expression of multiple DNA methylation transferases, which led to the differential expression of many genes. Analysis of differentially expressed genes in different treatments revealed that different treatments had gene specificity. However, the down-regulated GO pathway in all 4 treatments was involved in the negative regulation of the reproductive process, and post-embryonic development, and regulation of flower development. Several genes associated with DNA methylation and flowering regulation showed differential expression in response to various treatments.ConclusionsBoth DNA methylase reagent treatment and targeted silencing of the MET1 gene can cause differential expression of the genes. The operation of the exogenous application is simple, but the affected genes are exceedingly diverse and untargeted. Therefore, it is possible to construct populations with DNA methylation phenotypic diversity and to screen genes for DNA methylation regulation.