A Method to Study the Expression of DNA Methyltransferases in Aging Systems In Vitro

Numerous researches have focused on the genetic variations affecting SARS-CoV-2 infection, whereas the epigenetic effects are inadequately described. In this report, for the first time, we have identified potential candidate genes that might be regulated via SARS-CoV-2 induced DNA methylation changes in COVID-19 infection. At first, in silico transcriptomic data of COVID-19 lung autopsies were used to identify the top differentially expressed genes containing CpG Islands in their promoter region. Similar gene regulations were also observed in an in vitro model of SARS-CoV-2 infected lung epithelial cells (NHBE and A549). SARS-CoV-2 infection significantly decreased the levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in lung epithelial cells. Out of 14 candidate genes identified, the expression of 12 genes was upregulated suggesting promoter hypomethylation, while only two genes were downregulated suggesting promoter hypermethylation in COVID-19. Among those 12 upregulated genes, only HSPA1L and ULBP2 were found to be upregulated in AZA-treated lung epithelial cells and immune cells, suggesting their epigenetic regulation. To confirm the hypomethylation of these two genes during SARS-CoV-2 infection, their promoter methylation and mRNA expression levels were determined in the genomic DNA/RNA obtained from whole blood samples of asymptomatic, severe COVID-19 patients and equally matched healthy controls. The methylation level of HSPA1L was significantly decreased and the mRNA expression was increased in both asymptomatic and severe COVID-19 blood samples suggesting its epigenetic regulation by SARS-CoV-2 infection. Functionally, HSPA1L is known to facilitate host viral replication and has been proposed as a potential target for antiviral prophylaxis and treatment.

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Expression of DNA Methyltransferases Is Influenced by Growth Hormone in the Long-Living Ames Dwarf Mouse In Vivo and In Vitro

The Journals of Gerontology Series A ◽

10.1093/gerona/glt133 ◽

2013 ◽

Vol 69 (8) ◽

pp. 923-933 ◽

Cited By ~ 28

Author(s):

V. L. Armstrong ◽

S. Rakoczy ◽

L. Rojanathammanee ◽

H. M. Brown-Borg

Keyword(s):

Growth Hormone ◽

Dna Methyltransferases ◽

Dwarf Mouse ◽

Ames Dwarf ◽

Ames Dwarf Mouse

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Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

Microscopy and Microanalysis ◽

10.1017/s1431927618000016 ◽

2018 ◽

Vol 24 (1) ◽

pp. 29-37 ◽

Cited By ~ 4

Author(s):

Shuang Liang ◽

Zheng-Wen Nie ◽

Jing Guo ◽

Ying-Jie Niu ◽

Kyung-Tae Shin ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cellular Proliferation ◽

Dna Methyltransferases ◽

Blastocyst Stage ◽

Developmental Competence ◽

Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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Site-Specific DNA Demethylation as a Potential Target for Cancer Epigenetic Therapy

Epigenetics Insights ◽

10.1177/2516865720964808 ◽

2020 ◽

Vol 13 ◽

pp. 251686572096480

Author(s):

Sultan Abda Neja

Keyword(s):

Cpg Island ◽

Causal Effect ◽

Dna Methyltransferases ◽

Dna Demethylation ◽

Epigenetic Therapy ◽

In Vivo Model ◽

Transcription Activator ◽

Dna Hypermethylation ◽

Site Specific

Aberrant promoter DNA hypermethylation is a typical characteristic of cancer and it is often seen in malignancies. Recent studies showed that regulatory cis-elements found up-stream of many tumor suppressor gene promoter CpG island (CGI) attract DNA methyltransferases (DNMT) that hypermethylates and silence the genes. As epigenetic alterations are potentially reversible, they make attractive targets for therapeutic intervention. The currently used decitabine (DAC) and azacitidine (AZA) are DNMT inhibitors that follow the passive demethylation pathway. However, they lead to genome-wide demethylation of CpGs in cells, which makes difficult to use it for causal effect analysis and treatment of specific epimutations. Demethylation through specific demethylase enzymes is thus critical for epigenetic resetting of silenced genes and modified chromatins. Yet DNA-binding factors likely play a major role to guide the candidate demethylase enzymes upon its fusion. Before the advent of clustered regulatory interspaced short palindromic repeats (CRISPR), both zinc finger proteins (ZNFs) and transcription activator-like effector protein (TALEs) were used as binding platforms for ten-eleven translocation (TET) enzymes and both systems were able to induce transcription at targeted loci in an in vitro as well as in vivo model. Consequently, the development of site-specific and active demethylation molecular trackers becomes more than hypothetical to makes a big difference in the treatment of cancer in the future. This review is thus to recap the novel albeit distinct studies on the potential use of site-specific demethylation for the development of epigenetic based cancer therapy.

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DNA methylation and regulation of DNA methyltransferases in a freeze-tolerant vertebrate

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0091 ◽

2020 ◽

Vol 98 (2) ◽

pp. 145-153 ◽

Cited By ~ 2

Author(s):

Jing Zhang ◽

Liam J. Hawkins ◽

Kenneth B. Storey

Keyword(s):

Dna Methylation ◽

Transcriptional Repression ◽

Freeze Tolerance ◽

Dna Methyltransferases ◽

Wood Frog ◽

Activity Levels ◽

Wood Frogs ◽

Metabolic Role ◽

Freeze Tolerant

The wood frog is one of the few freeze-tolerance vertebrates. This is accomplished in part by the accumulation of cryoprotectant glucose, metabolic rate depression, and stress response activation. These may be achieved by mechanisms such as DNA methylation, which is typically associated with transcriptional repression. Hyperglycemia is also associated with modifications to epigenetic profiles, indicating an additional role that the high levels of glucose play in freeze tolerance. We sought to determine whether DNA methylation is affected during freezing exposure, and whether this is due to the wood frog’s response to hyperglycemia. We examined global DNA methylation and DNA methyltransferases (DNMTs) in the liver and muscle of frozen and glucose-loaded wood frogs. The results showed that levels of 5-methylcytosine (5mC) increased in the muscle, suggesting elevated DNA methylation during freezing. DNMT activities also decreased in muscle during thawing, glucose loading, and in vitro glucose experiments. Liver DNMT activities were similar to muscle; however, a varied response to DNMT levels and a decrease in 5mC highlight the metabolic role the liver plays during freezing. Glucose was also shown to decrease DNMT activity levels in the wood frog, in vitro, elucidating a potentially novel regulatory mechanism. Together these results suggest an interplay between freeze tolerance and hyperglycemic regulation of DNA methylation.

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Enzyme-mediated cytosine deamination by the bacterial methyltransferase M.MspI

Biochemical Journal ◽

10.1042/bj3320223 ◽

1998 ◽

Vol 332 (1) ◽

pp. 223-230 ◽

Cited By ~ 16

Author(s):

Jean-Marc ZINGG ◽

Jiang-Cheng SHEN ◽

Peter A. JONES

Keyword(s):

Dna Methyltransferases ◽

Amino Group ◽

Cytosine Deamination ◽

Physiological Conditions ◽

Solvent Water ◽

Naturally Occurring ◽

Haemophilus Parainfluenzae ◽

Hydrolytic Deamination

Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the reaction product S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.), causes very few cytosine deaminations, suggesting a mechanism in which M.MspI may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, sinefungin and 5´-amino-5´-deoxyadenosine, greatly increased the frequency of cytosine deamination mediated by M.MspI presumably by introducing a proton-donating amino group into the catalytic centre, thus facilitating the formation of an unstable enzyme–dihydrocytosine intermediate and hydrolytic deamination. Interestingly, two naturally occurring analogues, adenosine and 5´-methylthio-5´-deoxyadenosine, which do not contain a proton-donating amino group, also weakly increased the deamination frequency by M.MspI, even in the presence of AdoMet or AdoHcy. These analogues may trigger a conformational change in the enzyme without completely inhibiting the access of solvent water to the catalytic centre, thus allowing hydrolytic deamination of the enzyme–dihydrocytosine intermediate. Under normal physiological conditions the enzymes M.HpaII (from Haemophilus parainfluenzae), M.HhaI (from Haemophilus hemolytica) and M.MspI all increased the in vivo deamination frequency at the target cytosines with comparable efficiency.

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SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.648627 ◽

2021 ◽

Vol 9 ◽

Author(s):

Bo Jia ◽

Jun Chen ◽

Qin Wang ◽

Xiang Sun ◽

Jiusong Han ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Notch Signaling ◽

Osteogenic Differentiation ◽

Dna Methyltransferases ◽

Luciferase Reporter ◽

Calcium Deposition ◽

Alizarin Red

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

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A novel microRNA-based strategy to expand the differentiation potency of stem cells

10.1101/826446 ◽

2019 ◽

Author(s):

María Salazar-Roa ◽

Marianna Trakala ◽

Mónica Álvarez-Fernández ◽

Fátima Valdés-Mora ◽

Cuiqing Zhong ◽

...

Keyword(s):

Stem Cells ◽

De Novo ◽

Cardiac Injury ◽

Dna Methyltransferases ◽

Short Exposure ◽

Differentiation Potential ◽

Differentiation Capacity ◽

Tetraploid Complementation

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

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Antimutagenic, anti-inflammatory, and antioxidative activities of the juice of Vitis ficifolia var. Ganebu, a woody vine in the grape family, known as Ryukyu-ganebu in Japan

Genes and Environment ◽

10.1186/s41021-021-00225-y ◽

2021 ◽

Vol 43 (1) ◽

Author(s):

Sakae Arimoto-Kobayashi ◽

Ryoko Hida ◽

Nana Fujii ◽

Ryosuke Mochioka

Keyword(s):

Lipid Peroxidation ◽

Mutagenic Activity ◽

Dna Methyltransferases ◽

Significant Inhibition ◽

Prostaglandin E ◽

Active Components ◽

Chemopreventive Agents ◽

Anti Inflammatory

Abstract Background Mutation, inflammation, and oxidative damage including lipid-peroxidation are factors involved in the development of cancer. We investigated the antimutagenic, in vivo and in vitro anti-inflammatory, and antioxidative effects of the juice of Vitis ficifolia var. ganebu (known as Ryukyu-ganebu in Japan) harvested in Kuchinoshima island (hereafter, the juice is referred to as ganebu-K) in comparison with the juice of Vitis coignetiae (crimson glory vine, known as yamabudo in Japan; hereafter, the juice is referred to as yamabudo) which we found antimutagenic and anti-inflammatory effects. Results Ganebu-K inhibited the mutagenic activity of several carcinogens, MeIQx, IQ, Trp-P-2(NHOH), and MNNG, model compounds of tumor initiation. Using S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases, ganebu-K showed no significant inhibition of the mutagenicity of MNNG. Thus, DNA repair of O6-methylguanine produced by MNNG might be an antimutagenic target of the components in ganebu-K. Topical application of ganebu-K to the dorsal sides of mice resulted in potent suppression of acute edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Ganebu-K, but not yamabudo, exhibited significant inhibition of the induction of prostaglandin E2 (PGE2) induced by TPA. Components contained in ganebu-K, but not in yamabudo, might be responsible for the inhibition of the induction of PGE2. Ganebu-K inhibited in vivo lipid peroxidation and decreased the level of glutamic oxaloacetic transaminase induced by CCL4 treatment. Conclusions These results suggest that the active components in ganebu-K juice are not the same as those in yamabudo, and the components in ganebu-K are attractive candidates as chemopreventive agents.

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DNMT3b-mediated methylation of ZSWIM3 enhances inflammation in alcohol-induced liver injury via regulating TRAF2-mediated NF-κB pathway

Clinical Science ◽

10.1042/cs20200031 ◽

2020 ◽

Vol 134 (14) ◽

pp. 1935-1956 ◽

Cited By ~ 2

Author(s):

Hai-Di Li ◽

Xin Chen ◽

Jie-Jie Xu ◽

Xiao-Sa Du ◽

Yang Yang ◽

...

Keyword(s):

Liver Injury ◽

Zinc Finger ◽

Inflammatory Responses ◽

Tumor Necrosis Factor Receptor ◽

Dna Methyltransferases ◽

Aberrant Expression ◽

Ethanol Feeding ◽

And Function

Abstract The regulation of macrophages during inflammatory responses is a crucial process in alcoholic liver disease (ALD) and aberrant macrophage DNA methylation is associated with inflammation. Our preliminary screening results of macrophage methylation in the present study demonstrated the zinc finger SWI2/SNF2 and MuDR (SWIM)-domain containing 3 (ZSWIM3) were hypermethylated in the 5′ untranslated region (5′-UTR) region. ZSWIM3, a novel zinc finger-chelate domain of SWIM, is predicted to function in DNA-binding and protein-binding interactions. Its expression was found to be consistently decreased in macrophages isolated from livers of ethyl alcohol (EtOH)-fed mice and in EtOH+lipopolysaccharide (LPS)-induced RAW264.7 cells. Over-expression of ZSWIM3 was found to attenuate chronic+binge ethanol feeding-induced liver injury and inhibit inflammatory responses in vivo. Enforced expression of ZSWIM3 in vitro was also found to have anti-inflammatory effects. Aberrant expression of ZSWIM3 in alcohol-induced liver injury (ALI) was found to be associated with hypermethylation. Analysis of CpG prediction indicated the presence of two methylated sites in the ZSWIM3 promoter region and methylation inhibitor and DNA methyltransferases (DNMTs)-siRNA transfection were found to restore down-regulated ZSWIM3. Chromatin immunoprecipitation (ChIP) assay and molecular docking affirmed the role of DNMT 3b (DNMT3b) as a principal regulator of ZSWIM3 expression. Mechanistically, ZSWIM3 might affect inflammation by binding with tumor necrosis factor receptor-associated factor 2 (TRAF2), which further mediates the activation of the nuclear transcription factor κB (NF-κB) pathway. The present study, therefore, provides detailed insights into the possible structure and function of ZSWIM3 and thus, contributes new substantial research in the elucidation of the pathogenesis of ALI.

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