DNA Methyltransferases Contribute to Cold Tolerance in Ticks Dermacentor silvarum and Haemaphysalis longicornis (Acari: Ixodidae)

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum, and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days (P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly (P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts (P < 0.01), and mortality increased significantly (P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis. The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks.

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DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors

Cerebral Cortex ◽

10.1093/cercor/bhaa337 ◽

2020 ◽

Author(s):

Jing Wei ◽

Jia Cheng ◽

Nicholas J Waddell ◽

Zi-Jun Wang ◽

Xiaodong Pang ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Epigenetic Modification ◽

Substantial Reduction ◽

Dna Methyltransferases ◽

Male Rats ◽

Neural Pathways ◽

Dnmt Inhibitors ◽

Synaptic Functions

Abstract Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

Nucleic Acids Research ◽

10.1093/nar/gkaa938 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11495-11509

Author(s):

Michael Dukatz ◽

Sabrina Adam ◽

Mahamaya Biswal ◽

Jikui Song ◽

Pavel Bashtrykov ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Flanking Sequence ◽

Cpg Sites ◽

Binding Interface ◽

Target Sites ◽

Flanking Sequences

Abstract DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

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DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

Blood ◽

10.1182/blood-2011-06-357996 ◽

2011 ◽

Vol 118 (13) ◽

pp. 3559-3569 ◽

Cited By ~ 89

Author(s):

Rita Shaknovich ◽

Leandro Cerchietti ◽

Lucas Tsikitas ◽

Matthias Kormaksson ◽

Subhajyoti De ◽

...

Keyword(s):

Dna Methylation ◽

B Cells ◽

Germinal Center ◽

Dna Methyltransferase ◽

Cytosine Deaminase ◽

Dna Methyltransferases ◽

B Cell Differentiation ◽

Dual Roles ◽

Germinal Center B Cell ◽

Cellular Phenotypes

Abstract The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

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Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

PeerJ ◽

10.7717/peerj.4461 ◽

2018 ◽

Vol 6 ◽

pp. e4461 ◽

Cited By ~ 3

Author(s):

Jiang Li ◽

Caili Li ◽

Shanfa Lu

Keyword(s):

Dna Methylation ◽

Salicylic Acid ◽

Gene Family ◽

Sequence Alignment ◽

Dna Methyltransferase ◽

Epigenetic Modification ◽

Salvia Miltiorrhiza ◽

Dna Methyltransferases ◽

Genome Wide ◽

Wide Range

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants.

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A RID-like cytosine methyltransferase homologue controls sexual development in the fungusPodospora anserina

10.1101/575746 ◽

2019 ◽

Author(s):

P Grognet ◽

H Timpano ◽

F Carlier ◽

J Aït-Benkhali ◽

V Berteaux-Lecellier ◽

...

Keyword(s):

Dna Methylation ◽

Sexual Reproduction ◽

Sexual Development ◽

Genomic Dna ◽

Dna Methyltransferase ◽

Dna Methyltransferases ◽

Female Sterility ◽

Developmental Pathway ◽

Important Group ◽

Methylation Patterns

AbstractDNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detectedin vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any.To do so, we used the model ascomycete fungusP. anserina. We identified thePaRidgene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRiddefective mutants. Crosses involvingP. anserinaΔPaRidmutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRidbackground, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRidmutants with ectopic alleles ofPaRid, including GFP-tagged, point-mutated, inter-specific and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.Author SummarySexual reproduction is considered to be essential for long-term persistence of eukaryotic species. Sexual reproduction is controlled by strict mechanisms governing which haploids can fuse (mating) and which developmental paths the resulting zygote will follow. In mammals, differential genomic DNA methylation patterns of parental gametes, known as ‘DNA methylation imprints’ are essential to zygotic development, while in plants, global genomic demethylation often results in female-sterility. Although animal and fungi are evolutionary related, little is known about epigenetic regulation of gene expression and development in multicellular fungi. Here, we report on a gene of the model fungusPodospora anserina, encoding a protein called PaRid that looks like a DNA methyltrasferase. We showed that expression of the catalytically functional version of the PaRid protein is required in the maternal parental strain to form zygotes. By establishing the transcriptional profile ofPaRidmutant strain, we identified a set of PaRid direct and/or indirect target genes. Half of them are also targets of a mating-type transcription factor known to be a major regulator of sexual development. So far, there was no other example of identified RID targets shared with a well-known developmental pathway that is common to an important group of fungi, the Pezizomycotina

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Distinct Roles of Two DNA Methyltransferases from Cryphonectria parasitica in Fungal Virulence, Responses to Hypovirus Infection, and Viral Clearance

mBio ◽

10.1128/mbio.02890-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yo-Han Ko ◽

Kum-Kang So ◽

Jeesun Chun ◽

Dae-Hyuk Kim

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cryphonectria Parasitica ◽

Dna Methyltransferases ◽

Viral Clearance ◽

Biological Functions ◽

Fungal Virulence ◽

Content Type ◽

Genome Defense ◽

Fungal Dna

ABSTRACT Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations. IMPORTANCE Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus C. parasitica. We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance.

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LSH Cooperates with DNA Methyltransferases To Repress Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.01073-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 215-226 ◽

Cited By ~ 83

Author(s):

Kevin Myant ◽

Irina Stancheva

Keyword(s):

Dna Methylation ◽

Chromatin Remodeling ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Mammalian Cells ◽

Dna Methyltransferase ◽

Dna Methyltransferases ◽

Large Protein ◽

Large Protein Complex ◽

Nuclear Extracts

ABSTRACT LSH, a protein related to the SNF2 family of chromatin-remodeling ATPases, is required for efficient DNA methylation in mammals. How LSH functions to support DNA methylation and whether it associates with a large protein complex containing DNA methyltransferase (DNMT) enzymes is currently unclear. Here we show that, unlike many other chromatin-remodeling ATPases, native LSH is present mostly as a monomeric protein in nuclear extracts of mammalian cells and cannot be detected in a large multisubunit complex. However, when targeted to a promoter of a reporter gene, LSH acts as an efficient transcriptional repressor. Using this as an assay to identify proteins that are required for LSH-mediated repression we found that LSH cooperates with the DNMTs DNMT1 and DNMT3B and with the histone deacetylases (HDACs) HDAC1 and HDAC2 to silence transcription. We show that transcriptional repression by LSH and interactions with HDACs are lost in DNMT1 and DNMT3B knockout cells but that the enzymatic activities of DNMTs are not required for LSH-mediated silencing. Our data suggest that LSH serves as a recruiting factor for DNMTs and HDACs to establish transcriptionally repressive chromatin which is perhaps further stabilized by DNA methylation at targeted loci.

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iTRAQ-based quantitative proteomics analysis of cantaloupe (Cucumis melo var. saccharinus) after cold storage

10.21203/rs.2.20012/v2 ◽

2020 ◽

Author(s):

Wen Song ◽

Fengxian Tang ◽

Wenchao Cai ◽

Qin Zhang ◽

Fake Zhou ◽

...

Keyword(s):

Cold Stress ◽

Cold Tolerance ◽

Proteomic Analysis ◽

Molecular Mechanisms ◽

Treatment Time ◽

Chilling Injury ◽

Cold Treatment ◽

Cold Response ◽

Low Temperature Storage ◽

Metabolic Analysis

Abstract Background: During the low temperature storage, cantaloupe is susceptible to the cold stress, resulting in the loss of edible and commercial quality. To ascertain the molecular mechanisms of cold tolerance in cantaloupe, cold-sensitive cultivar Goldqueen-308 (GE) and cold-tolerant cultivar Jiashi-310 (JS) were used for quantitative proteomic analysis with iTRAQ in parallel. Results: In this work, two commercial cultivars were treated at 0.5℃ for 0, 12 and 24 days. Phenotypes assays showed that GE suffered a more severe damage as the cold treatment time extended. Proteomic analysis revealed that the number of differentially expressed proteins (DEPs) changed significantly over time in cold-exposed cantaloupe. Comparing with GE, JS responded in a prompter manner in terms of expressing cold-responding proteins during the similarly cold treatment. Furthermore, much more different groups of proteins were mobilized in response to the cold treatment in JS comparing with GE. Metabolic analysis indicated that more amino acids were up-regulated in JS during the early phases of cold stress. This study also identified some DEPs since they were up-regulated in JS or down-regulated in GE in terms of molecular mechanisms, which were mainly related to carbohydrate and energy metabolism, structure proteins, ROS scavening, amino acid metabolic and signaling transduction. Moreover, iTRAQ analysis was confirmed to be reliable via the results of phenotypes assays, metabolic analysis and q-PCR validation. Conclusion: By proteomics information,we found that the prompt response and the significant mobilization of proteins in JS maintained a higher level of cold tolerance, and the delay of cold response in GE could be a critical reason for the severe chilling injury. The candidate proteins we found will be the basis of future studies for further investigations and our findings may help to better understand the novel mechanisms of cold tolerance in cantaloupe.

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Selective Anchoring of DNA Methyltransferases 3A and 3B to Nucleosomes Containing Methylated DNA

Molecular and Cellular Biology ◽

10.1128/mcb.00484-09 ◽

2009 ◽

Vol 29 (19) ◽

pp. 5366-5376 ◽

Cited By ~ 145

Author(s):

Shinwu Jeong ◽

Gangning Liang ◽

Shikhar Sharma ◽

Joy C. Lin ◽

Si Ho Choi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Dna Methyltransferases ◽

Micrococcal Nuclease ◽

Nuclear Antigen ◽

Heterochromatin Protein 1 ◽

Histone Deacetylase 1 ◽

Methylation Patterns

ABSTRACT Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.

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