Function Of DNA Methyltransferases Research Articles

Using human disease mutations to understand de novo DNA methyltransferase function.

DNA methylation is a repressive epigenetic mark that is pervasive in mammalian genomes. It is deposited by DNA methyltransferase enzymes (DNMTs) that are canonically classified as having de novo (DNMT3A and DNMT3B) or maintenance (DNMT1) function. Mutations in DNMT3A and DNMT3B cause rare Mendelian diseases in humans and are cancer drivers. Mammalian DNMT3 methyltransferase activity is regulated by the non-catalytic region of the proteins which contain multiple chromatin reading domains responsible for DNMT3A and DNMT3B recruitment to the genome. Characterising disease-causing missense mutations has been central in dissecting the function and regulation of DNMT3A and DNMT3B. These observations have also motivated biochemical studies that provide the molecular details as to how human DNMT3A and DNMT3B mutations drive disorders. Here, we review progress in this area highlighting recent work that has begun dissecting the function of the disordered N-terminal regions of DNMT3A and DNMT3B. These studies have elucidated that the N-terminal regions of both proteins mediate novel chromatin recruitment pathways that are central in our understanding of human disease mechanisms. We also discuss how disease mutations affect DNMT3A and DNMT3B oligomerisation, a process that is poorly understood in the context of whole proteins in cells. This dissection of de novo DNMT function using disease-causing mutations provides a paradigm of how genetics and biochemistry can synergise to drive our understanding of the mechanisms through which chromatin misregulation causes human disease.

Emerging role of different DNA methyltransferases in the pathogenesis of cancer.

DNA methylation is one of the most essential epigenetic mechanisms to regulate gene expression. DNA methyltransferases (DNMTs) play a vital role in DNA methylation in the genome. In mammals, DNMTs act with some elements to regulate the dynamic DNA methylation patterns of embryonic and adult cells. Conversely, the aberrant function of DNMTs is frequently the hallmark in judging cancer, including total hypomethylation and partial hypermethylation of tumor suppressor genes (TSGs), which improve the malignancy of tumors, aggravate the ailment for patients, and significantly exacerbate the difficulty of cancer therapy. Since DNA methylation is reversible, currently, DNMTs are viewed as an important epigenetic target for drug development. However, the impression of DNMTs on cancers is still controversial, and therapeutic methods targeting DNMTs remain under exploration. This review mainly summarizes the relationship between the main DNMTs and cancers as well as regulatory mechanisms and clinical applications of DNMTs in cancer and highlights several forthcoming strategies for targeting DNMTs.

Role of DNMTs in the Brain.

DNA methyltransferases (DNMTs) are widely expressed in the brain, dictating the transcriptional activity of genes through various epigenetic mechanisms. Functional irregularities, alterations in the activity, and aberrant expression levels of DNMTs have been linked to various neurodevelopmental abnormalities, neuropsychiatric disorders, neurodegenerative diseases, and brain cancer. A continuously increasing number of studies address the roles DNMTs have in the brain, to reach a better understanding of their involvement in disease-related pathophysiologies, which in turn is required to dissect their applicability as potential therapeutic targets. This chapter provides an overview of DNMT function in the developing and the adult brain, putting a spotlight on their role in orchestrating diverse aspects of brain development, memory, and aging, followed by a discussion of associated neurodevelopmental and neurodegenerative disorders, and the implications in brain cancer.

Symposium Title: Application of Artificial Intelligence in Neuroscientific and Cognitive Studies

Aberrant DNA methylation has been implicated in the pathophysiology of several psychiatric disorders, including schizophrenia, depression, bipolar disorder, and posttraumatic stress disorder. DNA methylation deficits can potentially be corrected by the administration of drugs that inhibit the enzymes involved in DNA methylation called DNA methyltransferases (DNMTs). Several currently used psychoactive drugs have been shown to affect DNMT function and to be able to induce DNA demethylation, implying that epigenetic effects may contribute to their therapeutic efficacy. In addition, animal studies have shown that experimental use of DNMT inhibitors reverses gene expression and behavioral deficits that mimic endophenotypes associated with schizophrenia, depression, and anxiety. Although the prospect of using DNMT inhibitors in the treatment of the major psychiatric disorders is promising and exciting, significant basic and clinical research is necessary before these drugs are ready for clinical use.

Natural Products Impacting DNA Methyltransferases and Histone Deacetylases.

Epigenetics refers to heritable changes in gene expression and chromatin structure without change in a DNA sequence. Several epigenetic modifications and respective regulators have been reported. These include DNA methylation, chromatin remodeling, histone post-translational modifications, and non-coding RNAs. Emerging evidence has revealed that epigenetic dysregulations are involved in a wide range of diseases including cancers. Therefore, the reversible nature of epigenetic modifications concerning activation or inhibition of enzymes involved could be promising targets and useful tools for the elucidation of cellular and biological phenomena. In this review, emphasis is laid on natural products that inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) making them promising candidates for the development of lead structures for anticancer-drugs targeting epigenetic modifications. However, most of the natural products targeting HDAC and/or DNMT lack isoform selectivity, which is important for determining their potential use as therapeutic agents. Nevertheless, the structures presented in this review offer the well-founded basis that screening and chemical modifications of natural products will in future provide not only leads to the identification of more specific inhibitors with fewer side effects, but also important features for the elucidation of HDAC and DNMT function with respect to cancer treatment.

The Roles of DNA Methyltransferases 1 (DNMT1) in Regulating Sexual Dimorphism in the Cotton Mealybug, Phenacoccus solenopsis.

The cotton mealybug, Phenacoccus solenopsis, is an invasive pest that can cause massive damage to many host plants of agricultural importance. P. solenopsis is highly polyphagous, and shows extreme sexual dimorphism between males and females. The functions of DNA methyltransferase (DNMT) enzymes in the cotton mealybug have not been well studied. Here, we carried out an investigation of DNMTs in cotton mealybug to study their roles in sexual dimorphism. We found that the cotton mealybug has two copies of PsDnmt1, but Dnmt3 is absent. We then amplified the full-length cDNAs of PsDnmt1A (2225 bp) and PsDnmt1B (2862 bp) using rapid amplification cDNA ends (RACE). Quantitative reverse transcriptase PCR shows that both PsDnmt1A and PsDnmt1B are highly expressed in adult males, while the expression of PsDnmt1B is 30-fold higher in gravid females than in virgin females. We knocked down PsDnmt1A and PsDnmt1B with small interfering RNAs (siRNAs), and both genes were successfully down-regulated after 24 h or 72 h in adult females and pupa (t-test, p < 0.05). Down-regulating the expression of these two DNMT genes led to offspring lethality and abnormal body color in adult females. Furthermore, the silencing of PsDnmt1B induced abnormal wing development in emerged adult males. Our results provide evidence that PsDnmt1 plays a crucial role in regulating sexual dimorphism in the cotton mealybug.

Effects of SGI-1027 on Formation and Elimination of PrP^(Sc) in Prion-Infected Cells

Recently, SGI-1027, a well-known inhibitor of DNA-methyl transferases (DNMTs), was reported to effectively reduce formation of pathogenic PrP^(Sc) in prion-infected cells. Herein, we confirm the elimination of PrP^(Sc) in chronic wasting disease (CWD) prion-infected neurons by SGI-1027, and pinpoint the binding region of human prion protein to SGI-1027. SGI-1027 is broadly functional against various prion disease types, including human prions. Previously, the inhibitory effects of SGI-1027 on DNMT function is well tested in various cell culture models. While neither treatment with a DNMTs enhancer S-adenosyl-L-methionine (SAM), nor with their inhibitor, 5-azacytidine, prevented PrP^(Sc) propagation, SGI-1027 did. Our study suggest that the anti-prion effects of SGI-1027 are a result of its direct interaction with PrP^(C), which effectively interferes with the pathogenic conformational change of PrP^(C) to PrP^(Sc). We conclude that SGI-1027 driven suppression of pathogenic PrP^(Sc) is independent of DNMT.

DNA Methylation Status in Cancer Disease: Modulations by Plant-Derived Natural Compounds and Dietary Interventions

The modulation of the activity of DNA methyltransferases (DNMTs) represents a crucial epigenetic mechanism affecting gene expressions or DNA repair mechanisms in the cells. Aberrant modifications in the function of DNMTs are a fundamental event and part of the pathogenesis of human cancer. Phytochemicals, which are biosynthesized in plants in the form of secondary metabolites, represent an important source of biomolecules with pleiotropic effects and thus provide a wide range of possible clinical applications. It is well documented that phytochemicals demonstrate significant anticancer properties, and in this regard, rapid development within preclinical research is encouraging. Phytochemicals affect several epigenetic molecular mechanisms, including DNA methylation patterns such as the hypermethylation of tumor-suppressor genes and the global hypomethylation of oncogenes, that are specific cellular signs of cancer development and progression. This review will focus on the latest achievements in using plant-derived compounds and plant-based diets targeting epigenetic regulators and modulators of gene transcription in preclinical and clinical research in order to generate novel anticancer drugs as sensitizers for conventional therapy or compounds suitable for the chemoprevention clinical setting in at-risk individuals. In conclusion, indisputable anticancer activities of dietary phytochemicals linked with proper regulation of DNA methylation status have been described. However, precisely designed and well-controlled clinical studies are needed to confirm their beneficial epigenetic effects after long-term consumption in humans.

Critical function of DNA methyltransferase 1 in tomato development and regulation of the DNA methylome and transcriptome.

DNA methylation confers epigenetic regulation on gene expression and thereby on various biological processes. Tomato has emerged as an excellent system to study the function of DNA methylation in plant development. To date, regulation and function of DNA methylation maintenance remains unclear in tomato plants. Here, we report the critical function of tomato (Solanum lycopersicum) Methyltransferase 1 (SlMET1) in plant development and DNA methylome and transcriptome regulation. Using CRISPR-Cas9 gene editing, we generated slmet1 mutants and observed severe developmental defects with a frame-shift mutation, including small and curly leaves, defective inflorescence, and parthenocarpy. In leaf tissues, mutations in SlMET1 caused CG hypomethylation and CHH hypermethylation on a whole-genome scale, leading to a disturbed transcriptome including ectopic expression of many RIN target genes such as ACC2 in leaf tissues, which are normally expressed in fruits. Neither the CG hypomethylation nor CHH hypermethylation in the slmet1 mutants is related to tissue culture. Meanwhile, tissue culture induces non-CG hypomethylation, which occurs more frequently at gene regions than at TE regions. Our results depict SlMET1- and tissue culture-dependent tomato DNA methylomes, and that SlMET1 is required for maintaining a normal transcriptome and normal development of tomato.

Epigenetic regulation of B cell fate and function during an immune response.

The humoral immune response requires coordination of molecular programs to mediate differentiation into unique B cell subsets that help clear the infection and form immune memory. Epigenetic modifications are crucial for ensuring that the appropriate genes are transcribed or repressed during B cell differentiation. Recent studies have illuminated the changes in DNA methylation and histone post-translational modifications that accompany the formation of germinal center and antibody-secreting cells during an immune response. In particular, the B cell subset-specific expression and function of DNA methyltransferases and histone-modifying complexes that mediate epigenome changes have begun to be unravelled. This review will discuss the recent advances in this field, as well as highlight critical questions about the relationship between epigenetic regulation and B cell fate and function that are yet to be answered.

Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Characterization of SIRT1/DNMTs Functions and LINE-1 Methylation in Patients with Age-Related Macular Degeneration.

Previous studies proposed the application of DNA methylation signatures as clinical biomarkers of age-related macular degeneration (AMD). However, the characterization of Long Interspersed Nuclear Element-1 (LINE-1) methylation levels—a surrogate marker of global DNA methylation—in AMD patients has not been investigated so far. In the present study, we first characterized DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions in blood samples of 40 AMD patients and 10 age- and sex-matched controls. Then, we evaluated whether changes in DNMTs functions were associated with different LINE-1 methylation levels in leukocyte DNA. We demonstrated that total DNMTs activity was 48% higher in AMD patients than in controls (p = 0.005). AMD patients also exhibited up-regulation of DNMT1 and DNMT3B expression (FC = 2.6; p = 0.003 and FC = 2.4; p = 0.018, respectively). In line with increased DNMTs functions, the LINE-1 methylation level was higher in AMD patients than in controls (mean = 69.10%; SE = 0.68 vs. mean = 65.73%; SE = 0.59; p = 0.020). All p-values were adjusted by Bonferroni correction. In AMD patients, LINE-1 methylation level was positively associated with total DNMTs activity (r = 0.694; p < 0.001), DNMT1 (r = 0.579; p < 0.001), and DNMT3B (r = 0.521; p = 0.001) expression. Our results encourage further large-size prospective research to understand the relationship between LINE-1 methylation and AMD aetiology, and its usefulness in the clinical setting.

Mammalian DNA methyltransferases: new discoveries and open questions

As part of the epigenetic network, DNA methylation is a major regulator of chromatin structure and function. In mammals, it mainly occurs at palindromic CpG sites, but asymmetric methylation at non-CpG sites is also observed. Three enzymes are involved in the generation and maintenance of DNA methylation patterns. DNMT1 has high preference for hemimethylated CpG sites, and DNMT3A and DNMT3B equally methylate unmethylated and hemimethylated DNA, and also introduce non-CpG methylation. Here, we review recent observations and novel insights into the structure and function of mammalian DNMTs (DNA methyltransferases), including new structures of DNMT1 and DNMT3A, data on their mechanism, regulation by post-translational modifications and on the function of DNMTs in cells. In addition, we present news findings regarding the allosteric regulation and targeting of DNMTs by chromatin modifications and chromatin proteins. In combination, the recent publications summarized here impressively illustrate the intensity of ongoing research in this field. They provide a deeper understanding of key mechanistic properties of DNMTs, but they also document still unsolved issues, which need to be addressed in future research.

Resveratrol Modulates SIRT1 and DNMT Functions and Restores LINE-1 Methylation Levels in ARPE-19 Cells under Oxidative Stress and Inflammation.

The role of epigenetic alterations in the pathogenesis of retinal degenerative diseases, including age-related macular degeneration (AMD), has been pending so far. Our study investigated the effect of oxidative stress and inflammation on DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions, as well as on long interspersed nuclear element-1 (LINE-1) methylation, in human retinal pigment epithelial (ARPE-19) cells. Therefore, we evaluated whether treatment with resveratrol may modulate DNMT and SIRT1 functions and restore changes in LINE-1 methylation. Cells were treated with 25 mU/mL glucose oxidase (GOx) or 10 µg/mL lipopolysaccharide (LPS) to mimic oxidative or inflammatory conditions, respectively. Oxidative stress decreased DNMT1, DNMT3a, DNMT3b, and SIRT1 expression (p-values < 0.05), as well as total DNMTs (−28.5%; p < 0.0001) and SIRT1 (−29.0%; p < 0.0001) activities. Similarly, inflammatory condition decreased DNMT1 and SIRT1 expression (p-values < 0.05), as well as total DNMTs (−14.9%; p = 0.007) and SIRT1 (−20.1%; p < 0.002) activities. Interestingly, GOx- and LPS-treated cells exhibited lower LINE-1 methylation compared to controls (p-values < 0.001). We also demonstrated that treatment with 10 μM resveratrol for 24 h counteracted the detrimental effect on DNMT and SIRT1 functions, and LINE-1 methylation, in cells under oxidative and inflammatory conditions. However, further studies should explore the perspectives of resveratrol as a suitable strategy for the prevention and/or treatment of retinal degenerative diseases.

Curcumin Modulates DNA Methyltransferase Functions in a Cellular Model of Diabetic Retinopathy.

Hyperglycaemia-induced oxidative stress appears to be involved in the aetiology of diabetic retinopathy (DR), a major public health issue, via altering DNA methylation process. We investigated the effect of hyperglycaemia on retinal DNA methyltransferase (DNMT) expression in diabetic mice, using Gene Expression Omnibus datasets. We also evaluated the effect of curcumin both on high glucose-induced reactive oxygen species (ROS) production and altered DNMT functions, in a cellular model of DR. We observed that three months of hyperglycaemia, in insulin-deficient Ins2Akita mice, decrease DNMT1 and DNMT3a expression levels. In retinal pigment epithelium (RPE) cells, we also demonstrated that high glucose-induced ROS production precedes upregulation of DNMT expression and activity, suggesting that changes in DNMT function could be mediated by oxidative stress via a potential dual effect. The early effect results in decreased DNMT activity, accompanied by the highest ROS production, while long-term oxidative stress increases DNMT activity and DNMT1 expression. Interestingly, treatment with 25 μM curcumin for 6 hours restores ROS production, as well as DNMT functions, altered by the exposure of RPE to acute and chronic high glucose concentration. Our study suggests that curcumin may represent an effective antioxidant compound against DR, via restoring oxidative stress and DNMT functions, though further studies are recommended.

Host Methyltransferases and Demethylases: Potential New Epigenetic Targets for HIV Cure Strategies and Beyond.

A successful HIV cure strategy may require reversing HIV latency to purge hidden viral reservoirs or enhancing HIV latency to permanently silence HIV transcription. Epigenetic modifying agents show promise as antilatency therapeutics in vitro and ex vivo, but also affect other steps in the viral life cycle. In this review, we summarize what we know about cellular DNA and protein methyltransferases (PMTs) as well as demethylases involved in HIV infection. We describe the biology and function of DNA methyltransferases, and their controversial role in HIV infection. We further explain the biology of PMTs and their effects on lysine and arginine methylation of histone and nonhistone proteins. We end with a focus on protein demethylases, their unique modes of action and their emerging influence on HIV infection. An outlook on the use of methylation-modifying agents in investigational HIV cure strategies is provided.

An association between overexpression of DNA methyltransferase 3B4 and clear cell renal cell carcinoma.

It is well known that abnormal DNA methylations occur frequently in kidney cancer. However, it remains unclear exactly which types of DNA methyltransferases (DNMT) contribute to the pathologies of kidney cancers. In order to determine the functions of DNA methyltransferase in kidney tumorigenesis on the molecular level, we examined the mRNA expression levels of DNMT1, DNMT3A, DNMT3B, and DNMT3B variants in renal cell carcinoma tissue. Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were increased in renal cell carcinoma tissue compared with adjacent control tissues. Additionally, Alu elements and long interspersed nuclear elements (LINE-1) were hypomethylated in renal cell carcinoma tissue. Meanwhile, methylation of the promoter for RASSF1A, a tumor suppressor gene, was moderately increased in renal cell carcinoma tissue, while RASSF1A expression was decreased. Thus, our data suggest that the overexpression of DNMT3B4 may play an important role in human kidney tumorigenesis through chromosomal instability and methylation of RASSF1A.

DNA Methylation Biphasically Regulates 3T3-L1 Preadipocyte Differentiation.

Better understanding the mechanisms underlying adipogenesis may provide novel therapeutic targets in the treatment of obesity. Most studies investigating the mechanisms underlying adipogenesis focus on highly regulated transcriptional pathways; little is known about the epigenetic mechanisms in this process. Here, we determined the role of DNA methylation in regulating 3T3-L1 adipogenesis in early and late stage of differentiation. We found that inhibiting DNA methylation pharmacologically by 5-aza-2'-deoxycytidine (5-aza-dC) at early stage of 3T3-L1 differentiation markedly suppressed adipogenesis. This inhibition of adipogenesis by 5-aza-dC was associated with up-regulation of Wnt10a, an antiadipogenic factor, and down-regulation of Wnt10a promoter methylation. In contrast, inhibiting DNA methylation by 5-aza-dC at late stage of differentiation enhanced the lipogenic program. The differential effects of 5-aza-dC on adipogenesis were confirmed by gain or loss of function of DNA methyltransferase 1 using genetic approaches. We further explored the molecular mechanism underlying the enhanced lipogenesis by inhibition of DNA methylation at late stage of differentiation. The Srebp1c promoter is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNA methyltransferase 1 bound to the methylation region at the Srebp1c promoter. Pyrosequencing analysis revealed that the DNA methylation at the key cis-elements of the Srebp1c promoter was down-regulated in adipogenesis. Further, luciferase reporter assays showed that the Srebp1c promoter activity was dramatically up-regulated by the unmethylated promoter compared with the fully methylated promoter. Thus DNA methylation appears to exert a biphasic regulatory role in adipogenesis, promoting differentiation at early stage while inhibiting lipogenesis at late stage of 3T3-L1 preadipocyte differentiation.

Aberrant expression and localization of deoxyribonucleic acid methyltransferase 3B in endometriotic stromal cells

To define the expression and function of DNA methyltransferases (DNMTs) in response to decidualizing stimuli in endometriotic cells compared with healthy endometrial stroma. Basic science. University research center. Premenopausal women with or without endometriosis. Primary cultures of stromal cells from healthy endometrium (E-IUM) or endometriomas (E-OSIS) were subjected to in vitro decidualization (IVD) using 1 μM medroxyprogesterone acetate, 35 nM 17β-estradiol, and 0.05 mM 8-Br-cAMP. Expression of DNMT1, DNMT3A, and DNMT3B in E-IUM and E-OSIS were assessed by quantitative real-time polymerase chain reaction and immunoblotting. Recruitment of DNMT3B to the promoters of steroidogenic factor 1 (SF-1) and estrogen receptor α (ESR1) was examined by chromatin immunoprecipitation. IVD treatment reduced DNMT3B messenger RNA (74%) and protein levels (81%) only in E-IUM; DNMT1 and DNMT3A were unchanged in both cell types. Significantly more DNMT3B bound to the SF-1 promoter in E-IUM compared with E-OSIS, and IVD treatment reduced binding in E-IUM to levels similar to those in E-OSIS. Enrichment of DNMT3B across 3 ESR1 promoters was reduced in E-IUM after IVD, although the more-distal promoter showed increased DNMT3B enrichment in E-OSIS after IVD. The inability to downregulate DNMT3B expression in E-OSIS may contribute to an aberrant epigenetic fingerprint that misdirects gene expression in endometriosis and contributes to its altered response to steroid hormones.

Hepatitis viruses exploitation of host DNA methyltransferases functions.

Hepatitis B virus (HBV), hepatitis C virus (HCV) and Delta (HDV) infections are a global health burden. With different routes of infection and biology, HBV, HCV and HDV are capable to induce liver cirrhosis and cancer by impinging on epigenetic mechanisms altering host cell's pathways. In the present manuscript, we reviewed the published studies taking into account the relationship between the hepatitis viruses and the DNA methyltransferases proteins.