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

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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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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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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DNA Methylation Profiling of Pediatric AML Reveals That Hypomethylation of MN1 Is Characteristic of Inv(16) AML and a Driver of MN1 Overexpression

Blood ◽

10.1182/blood.v124.21.867.867 ◽

2014 ◽

Vol 124 (21) ◽

pp. 867-867

Author(s):

Nicole S.D. Larmonie ◽

Marry M. van den Heuvel-Eibrink ◽

Askar Obulkasim ◽

Valerie de Haas ◽

Dirk Reinhardt ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cpg Island ◽

Dna Methyltransferases ◽

Genetic Abnormalities ◽

Pediatric Aml ◽

Methylation Patterns ◽

Dnmt3b Expression ◽

Methylation Profiling ◽

Epigenetic Processes

Abstract Primary refractory and relapsed pediatric acute myeloid leukemia (AML) still lead to a significant number of childhood cancer deaths, despite the current chemotherapeutic regimens. AML leukemogenesis is driven by collaborative genetic abnormalities that induce hematopoietic maturation arrest and cell proliferation. Particular AML-associated maturation inhibiting aberrations are known to target chromatin regulators, thus directly influencing the transcriptional program of leukemic cells. Therapies targeting epigenetic processes, e.g. with hypomethylation-inducing agents, are therefore becoming an attractive therapeutic strategy in adult AML. AML biology in children is not equivalent to that of adults, thus methylation patterns seen in adult AML cannot be extrapolated to pediatric AML. Therefore there is a need to unravel the mechanism behind changes in epigenetic processes as the result of AML-causing genetic abnormalities in order to develop new drugs for pediatric AML. We hypothesized that pediatric AML samples have distinct DNA-methylation patterns which may provide a rationale for treatment with demethylating agents in specific pediatric AML subtypes. Furthermore, these differences in methylation could be characteristic for AML subgroups and that particular methylation patterns drive the expression of specific genes which may play a key role in the tumorigenesis of these AML leukemias. We performed genome-wide CpG-island methylation profiling on a representative and molecularly characterized cohort of pediatric patients with de novo AML. Empirical Bayes Wilcoxon rank-sum test showed that AML patients carrying inv(16)(p13;q22) (n=9) have distinct DNA methylation patterns when compared to non-inv(16) AML patients (n=143) (consisting mainly of MLL-rearranged, t(8;21), t(15;17), t(8;16) AML and AML cases with a normal karyotype). The MN1 gene ranked as most significantly differentially methylated in inv(16) AML compared to non-inv(16) AML, with inv(16) AML cases having significantly (p=2x10-6) lower methylation levels compared to non-inv(16) AML cases. Hypomethylation of specific regions of the MN1-associated CpG-island was confirmed by methylation specific PCR and bisulfite sequencing. Subsequent gene expression (GEP) data on 294 pediatric AML patients showed that MN1 was 8 fold higher expressed in patients carrying inv(16) compared to all other patients (9.9, n=35 vs 6.9, n=259, p<0.001). Furthermore, integrating GEP and methylation array data showed that MN1 expression negatively correlated (ρs= 0.82, p=0.011) with methylation levels, which is in agreement with the biological assumption of methylation and gene expression. Since genes known to regulate DNA methylation have frequently been shown to be mutated in adult AML we determined whether a decreased expression of DNA methyltransferases, DNMT1, DNMT2, DNMT3A, DNMT3B, could be the cause of a hypometylated MN1 locus in inv(16) AML. Our findings show that only DNMT3B expression was significantly (p=8x10-15) lower in inv(16) cases compared to non-inv(16) cases. To test whether hypomethylation of the MN1 CpG-island and the overexpression of MN1 is the result of decreased DNMT1 expression, HL60 cells which express negligible levels of MN1 were treated with the DNMT1 inhibitor Decitabine. This showed that treatment of HL60 cells with Decitabine led to increase of MN1 transcript levels, however, not as high as those observed in patient samples. This suggests that DNMT1 activity may not be the only DNA methyltransferase influencing expression of MN1 in inv(16) patients. Interestingly, we observed a high (ρs= 0.42) correlation between MN1 methylation and DNMT3B expression, which suggests DNMT3B could be an important DNA methyltransferase involved in regulating MN1expression. Overall we show that pediatric AML patients carrying and inv(16) have a characteristic DNA methylation pattern compared to other AML patients carrying specific cytogenetic aberrations. Furthermore, our data suggest that hypomethylation of the MN1 gene is an underlying mechanism for high MN1 expression in inv(16)(p13;q22) patients possibly regulated by multiple DNA methyltransferases. Disclosures No relevant conflicts of interest to declare.

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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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Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.40 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jessilyn Dunn ◽

Haiwei Qiu ◽

Soyeon Kim ◽

Daudi Jjingo ◽

Ryan Hoffman ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Methyltransferase ◽

Methylation Status ◽

Western Diet ◽

Dependent Manner ◽

Gene Promoters ◽

Genome Wide ◽

Methylation Patterns ◽

Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

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Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Endocrinology ◽

10.1210/en.2009-0499 ◽

2009 ◽

Vol 150 (10) ◽

pp. 4681-4691 ◽

Cited By ~ 91

Author(s):

Aparna Mahakali Zama ◽

Mehmet Uzumcu

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Ovarian Function ◽

Endocrine Disrupting Chemicals ◽

Neonatal Exposure ◽

Dna Hypermethylation ◽

Promoter Regions ◽

Altered Expression ◽

Arbitrarily Primed Pcr ◽

Methylation Patterns

Abstract Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ERα was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 μg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERα and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ERα promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

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Stochastic Modeling Reveals Kinetic Heterogeneity in Post-replication DNA Methylation

10.1101/677013 ◽

2019 ◽

Author(s):

Luis Busto-Moner ◽

Julien Morival ◽

Arjang Fahim ◽

Zachary Reitz ◽

Timothy L. Downing ◽

...

Keyword(s):

Mathematical Modeling ◽

Dna Methylation ◽

Rate Constants ◽

Cytosine Methylation ◽

Temporal Dynamics ◽

Epigenetic Modification ◽

Embryonic Stem ◽

Likelihood Estimation ◽

Dna Methyltransferases ◽

Methylation Patterns

AbstractDNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.

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DNA Methylation Patterns in Saliva of Tobacco users with High-Risk Oral Potentially Malignant Disorders

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i32b31754 ◽

2021 ◽

pp. 129-137

Author(s):

Mimansha Patel ◽

Madhuri Nitin Gawande ◽

Minal Shashikant Chaudhary ◽

Alka Harish Hande

Keyword(s):

Dna Methylation ◽

High Risk ◽

Malignant Transformation ◽

Promoter Methylation ◽

Genomic Dna ◽

Premalignant Lesions ◽

Oral Potentially Malignant Disorders ◽

Potentially Malignant Disorders ◽

Potentially Malignant ◽

Methylation Patterns

Background: “Oral Potentially Malignant Disorder (OPMD)” is a well-known symptom that, if untreated, can be carcinogenic. It includes leukoplakia, erythroplakia or erythroleukoplakia. One of the typical premalignant lesions of the oral cavity is “oral leukoplakias (OLs),” which frequently precedes “OSCCs.”OLs with dysplastic characteristics are considered to be at a higher risk of “malignant transformation.” So, early diagnosis of "oral squamous cell carcinomas (OSCCs)" is desperately required to enhance patient prognosis and quality of life (QOL).As a result, we examined the distinctive promoter methylation presence in high-risk OLs. Objectives: To detect, compare & correlate “DNA methylation” patterns in normal individuals, tobacco users without disease and tobacco users with the disease. Methodology: With the participants' full consent, 48 saliva samples were obtained and prepared. DNA isolation, restriction digestion of genomic DNA, extraction of restriction enzyme digested genomic DNA, Polymerase Chain Reaction (PCR), and Agarose Gel Electrophoresis (AGE) were all carried out. Expected results: This study will help us to assess the use of Saliva as an aid to identifying both high and low risk “Oral Potentially Malignant Disorders.” Conclusion: Peculiar promoter methylation of various genes was related to a high possibility of malignant transformation in OLs.

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Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction

eLife ◽

10.7554/elife.47859 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Shir Toubiana ◽

Miriam Gagliardi ◽

Mariarosaria Papa ◽

Roberta Manco ◽

Maty Tzukerman ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Pharmacological Intervention ◽

Icf Syndrome ◽

Genome Wide ◽

Mammalian Genomes ◽

Induced Pluripotent ◽

Methylation Patterns

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the ICF1 telomeric phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction.

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Detection of DNA methyltransferase activity using allosteric molecular beacons

The Analyst ◽

10.1039/c5an01763g ◽

2016 ◽

Vol 141 (2) ◽

pp. 579-584 ◽

Cited By ~ 9

Author(s):

Weiting Zhang ◽

Xiaolong Zu ◽

Yanling Song ◽

Zhi Zhu ◽

Chaoyong James Yang

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Molecular Beacon ◽

Molecular Beacons ◽

Sensitive Detection ◽

Methyltransferase Activity ◽

Mtase Activity ◽

Adenine Methylation ◽

Methylation Patterns

Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity.

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