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Methods (San Diego, Calif.) Mar 2021Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant... (Review)
Review
Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant methylation has been associated to the majority of the diseases, including cancer, neurodegenerative, cardiovascular and autoimmune disorders. Analysis of DNA methylation patterns is crucial to understand the underlying molecular mechanism of these diseases. Moreover, DNA methylation patterns could be used as biomarker for clinical management, such as diagnosis, prognosis and treatment response. Nowadays, a variety of high throughput methods for DNA methylation have been developed to analyze the methylation status of a high number of CpGs at once or even the whole genome. However, identification of specific methylation patterns at specific loci is essential for validation and also as a tool for diagnosis. In this review, we describe the most commonly used approaches to evaluate specific DNA methylation. There are three main groups of techniques that allow the identification of specific regions that are differentially methylated: bisulfite conversion-based methods, restriction enzyme-based approaches, and affinity enrichment-based assays. In the first group, specific restriction enzymes recognize and cleave unmethylated DNA, leaving methylated sequences intact. Bisulfite conversion methods are the most popular approach to distinguish methylated and unmethylated DNA. Unmethylated cytosines are deaminated to uracil by sodium bisulfite treatment, while the methyl cytosines remain unconverted. In the last group, proteins with methylation binding domains or antibodies against methyl cytosines are used to recognize methylated DNA. In this review, we provide the theoretical basis and the framework of each technique as well as the analysis of their strength and the weaknesses.
Topics: Aging; CpG Islands; DNA Methylation; Epigenesis, Genetic; Epigenomics; Neoplasms; Obesity; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 32640317
DOI: 10.1016/j.ymeth.2020.06.021 -
Biochemical Genetics Aug 2022If genetics defines the inheritance of DNA, epigenetics aims to regulate and make it adaptable. Epigenetic alterations include DNA methylation, chromatin remodelling,... (Review)
Review
If genetics defines the inheritance of DNA, epigenetics aims to regulate and make it adaptable. Epigenetic alterations include DNA methylation, chromatin remodelling, post-translational modifications of histone proteins and activity of non-coding RNAs. Several studies, especially in animal models, have reported transgenerational inheritance of epigenetic marks. However, evidence of transgenerational inheritance in humans via germline in the absence of any direct exposure to the driving external stimulus remains controversial. Most of the epimutations exist in relation with genetic variants. The present review looks at intergenerational and transgenerational inheritance in humans, (both father and mother) in response to diet, exposure to chemicals, stress, exercise, and disease status. If not transgenerational, at least intergenerational human studies could help to understand early processes of inheritance. In humans, female and male germline development follow separate paths of epigenetic events and both oocyte and sperm possess their own unique epigenomes. While DNA methylation alterations are reset during epigenetic reprogramming, non-coding RNAs via human sperm provide evidence of being reliable carriers for transgenerational inheritance. Human studies reveal that one mechanism of epigenetic inheritance cannot be applied to the complete human genome. Multiple factors including time, type, and tissue of exposure determine if the modified epigenetic mark could be transmissible and till which generation. Population-specific differences should also be taken into consideration while associating inheritance to an environmental exposure. A longitudinal study targeting one environmental factor, but different population groups should be conducted at a specific geographical location to pinpoint heritable epigenetic changes.
Topics: Animals; DNA Methylation; Epigenesis, Genetic; Female; Humans; Inheritance Patterns; Longitudinal Studies; Male; Semen
PubMed: 34792705
DOI: 10.1007/s10528-021-10155-7 -
Gene Jul 2023DNA methylation is one of the epigenetic modifications of the genome, the essence of which is the attachment of a methyl group to nitrogenous bases. In the eukaryote... (Review)
Review
DNA methylation is one of the epigenetic modifications of the genome, the essence of which is the attachment of a methyl group to nitrogenous bases. In the eukaryote genome, cytosine is methylated in the vast majority of cases. About 98% of cytosines are methylated as part of CpG dinucleotides. They, in turn, form CpG islands, which are clusters of these dinucleotides. Islands located in the regulatory elements of genes are in particular interest. They are assumed to play an important role in the regulation of gene expression in humans. Besides that, cytosine methylation serves the functions of genomic imprinting, transposon suppression, epigenetic memory maintenance, X- chromosome inactivation, and embryonic development. Of particular interest are the enzymatic processes of methylation and demethylation. The methylation process always depends on the work of enzymatic complexes and is very precisely regulated. The methylation process largely depends on the functioning of three groups of enzymes: writers, readers and erasers. Writers include proteins of the DNMT family, readers are proteins containing the MBD, BTB/POZ or SET- and RING-associated domains and erasers are proteins of the TET family. Whereas demethylation can be performed not only by enzymatic complexes, but also passively during DNA replication. Hence, the maintenance of DNA methylation is important. Changes in methylation patterns are observed during embryonic development, aging, and cancers. In both aging and cancer, massive hypomethylation of the genome with local hypermethylation is observed. In this review, we will review the current understanding of the mechanisms of DNA methylation and demethylation in humans, the structure and distribution of CpG islands, the role of methylation in the regulation of gene expression, embryogenesis, aging, and cancer development.
Topics: Humans; CpG Islands; DNA Methylation; Neoplasms; Physiological Phenomena; Gene Expression Regulation, Neoplastic; Transcription, Genetic
PubMed: 37211289
DOI: 10.1016/j.gene.2023.147487 -
Dental Materials : Official Publication... May 2021The stability of the bond between polymeric adhesives to mineralized substrates is crucial in many biomedical applications. The objective of this study was to determine...
UNLABELLED
The stability of the bond between polymeric adhesives to mineralized substrates is crucial in many biomedical applications. The objective of this study was to determine the effect of methyl substitution at the α- and β-carbons on the kinetics of polymerization, monomer hydrolytic stability, and long-term bond strength to dentin for methacrylamide- and methacrylate-based crosslinked networks for dental adhesive applications.
METHODS
Secondary methacrylamides (α-CH substituted=1-methyl HEMAM, β-CH substituted=2-methyl HEMAM, and unsubstituted=HEMAM) and OH-terminated methacrylates (α- and β-CH mixture=1-methyl HEMA and 2-methyl HEMA, and unsubstituted=HEMA) were copolymerized with urethane dimethacrylate. The kinetics of photopolymerization were followed in real-time using near-IR spectroscopy. Monomer hydrolysis kinetics were followed by NMR spectroscopy in water at pH 1 over 30 days. Solvated adhesives (40 vol% ethanol) were used to bond composite to dentin and microtensile bond strength (μTBS) measured after 24h and 6 months storage in water at 37°C.
RESULTS
The rate of polymerization increased in the following order: OH-terminated methacrylates≥methacrylamides>NH-terminated methacrylates, with minimal effect of the substitution. Final conversion ranged between 79% for 1-methyl AEMA and 94% for HEMA. 1-methyl-HEMAM showed the highest and most stable μTBS, while HEMA showed a 37% reduction after six months All groups showed measurable degradation after up to 4 days in pH 1, with the methacrylamides showing less degradation than the methacrylates. Additionally, transesterification products were observed in the methacrylamide groups.
SIGNIFICANCE
Amide monomers were significantly more stable to hydrolysis than the analogous methacrylates. The addition of a α- or β-CH groups increased the rate of hydrolysis, with the magnitude of the effect tracking with the expected base-catalyzed hydrolysis of esters or amides, but opposite in influence. The α-CH substituted secondary methacrylamide, 1-methyl HEMAM, showed the most stable adhesive interface. A side reaction was observed with transesterification of the monomers studied under ambient conditions, which was not expected under the relatively mild conditions used here, which warrants further investigation.
Topics: Acrylamides; Composite Resins; Dental Bonding; Dental Cements; Dentin; Dentin-Bonding Agents; Materials Testing; Methacrylates; Methylation; Resin Cements; Tensile Strength
PubMed: 33663882
DOI: 10.1016/j.dental.2021.02.004 -
Frontiers in Immunology 2022Type 1 diabetes mellitus (T1DM) is caused by immune cell-mediated β-cell dysfunction. In recent decades, N6-methyladenosine (m6A) has attracted widespread attention in...
BACKGROUND
Type 1 diabetes mellitus (T1DM) is caused by immune cell-mediated β-cell dysfunction. In recent decades, N6-methyladenosine (m6A) has attracted widespread attention in the scientific research field because it plays vital roles in the pathogenesis of immunity-related diseases, including autoimmune diseases. However, neither the m6A modification profile nor the potential role it plays in T1DM pathogenesis has been investigated to date.
MATERIALS AND METHODS
An m6A mRNA epitranscriptomic microarray analysis was performed to analyze m6A regulator expression patterns and m6A methylation patterns in immune cells of T1DM patients (n=6) and healthy individuals (n=6). A bioinformatics analysis was subsequently performed to explore the potential biological functions and signaling pathways underlying T1DM pathogenesis. Furthermore, mRNA expression and m6A methylation levels were subsequently verified by qRT-PCR and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), respectively, in the T1DM and healthy groups (n=6 per group).
RESULTS
Among the multiple m6A regulators, METTL3 and IGF2BP2 had significantly downregulated expression, and YTHDC1 and HNRNPA2B1 had significantly upregulated expression in the T1DM group relative to the healthy group. The microarray analysis revealed 4247 differentially methylated transcripts, including 932 hypermethylated and 3315 hypomethylated transcripts, and 4264 differentially expressed transcripts, including 1818 upregulated transcripts and 2446 downregulated transcripts in the T1DM group relative to the healthy group. An association analysis between methylation and gene expression demonstrated that the expression of 590 hypermethylated transcripts was upregulated, and that of 1890 hypomethylated transcripts was downregulated. Pearson correlation analysis showed significant correlations between the expression levels of differentially expressed m6A regulators and the methylation levels of differentially methylated transcripts and significant correlations between the expression levels of differentially expressed m6A regulators and that of differentially expressed transcripts. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that differentially methylated transcripts were involved in pathways related to immunity, including some closely associated with T1DM.
CONCLUSIONS
Our study presents m6A regulator expression patterns and m6A methylation patterns of immune cells in T1DM, showing that the m6A mark and m6A regulators are promising targets for T1DM diagnosis and treatment.
Topics: Humans; Methylation; Diabetes Mellitus, Type 1; Protein Processing, Post-Translational; Autoimmune Diseases; Gene Ontology; Methyltransferases; RNA-Binding Proteins
PubMed: 36457997
DOI: 10.3389/fimmu.2022.1030728 -
Journal of Molecular Graphics &... May 2021Methyl transfer reactions, mediated by methyltransferases (MeTrs), such as methionine synthase (MetH) or monomethylamine: CoM (MtmBC), constitute one of the most...
Methyl transfer reactions, mediated by methyltransferases (MeTrs), such as methionine synthase (MetH) or monomethylamine: CoM (MtmBC), constitute one of the most important classes of vitamin B-dependent reactions. The challenge in exploring the catalytic function of MeTrs is related to their modular structure. From the crystallographic point of view, the structure of each subunit has been determined, but there is a lack of understanding of how each subunit interacts with each other. So far, theoretical studies of methyl group transfer were carried out for the structural models of the active site of each subunit. However, those studies do not include the effect of the enzymatic environment, which is crucial for a comprehensive understanding of enzyme-mediated methyl transfer reactions. Herein, to explore how two subunits interact with each other and how the methyl transfer reaction is catalyzed by MeTrs, molecular docking of the functional units of MetH and MtmBC was carried out. Along with the interactions of the functional units, the reaction coordinates, including the Co-C bond distance for methylation of cob(I)alamin (CoCbl) and the C-S bond distance in demethylation reaction of cob(III)alamin (CoCbl), were considered. The functional groups should be arranged so that there is an appropriate distance to transfer a methyl group and present results indicate that steric interactions can limit the number of potential arrangements. This calls into question the possibility of S2-type mechanism previously proposed for MeTrs. Further, it leads to the conclusion that the methyl transfer reaction involves some spatial changes of modules suggesting an alternate radical-based pathway for MeTrs-mediated methyl transfer reactions. The calculations also showed that changes in torsion angles induce a change in reaction coordinates, namely Co-C and C-S bond distances, for the methylation and demethylation reactions catalyzed both by MetH and MtmBC.
Topics: Catalysis; Methylation; Methyltransferases; Molecular Docking Simulation; Vitamin B 12
PubMed: 33529932
DOI: 10.1016/j.jmgm.2020.107831 -
Environmental Pollution (Barking, Essex... Aug 2023The cell surface adsorption and intracellular uptake of mercuric mercury Hg(II) and methylmercury (MeHg) are important in determining the fate and transformation of Hg...
The cell surface adsorption and intracellular uptake of mercuric mercury Hg(II) and methylmercury (MeHg) are important in determining the fate and transformation of Hg in the environment. However, current information is limited about their interactions with two important groups of microorganisms, i.e., methanotrophs and Hg(II)-methylating bacteria, in aquatic systems. This study investigated the adsorption and uptake dynamics of Hg(II) and MeHg by three strains of methanotrophs, Methylomonas sp. strain EFPC3, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath, and two Hg(II)-methylating bacteria, Pseudodesulfovibrio mercurii ND132 and Geobacter sulfurreducens PCA. Distinctive behaviors of these microorganisms towards Hg(II) and MeHg adsorption and intracellular uptake were observed. The methanotrophs took up 55-80% of inorganic Hg(II) inside cells after 24 h incubation, lower than methylating bacteria (>90%). Approximately 80-95% of MeHg was rapidly taken up by all the tested methanotrophs within 24 h. In contrast, after the same time, G. sulfurreducens PCA adsorbed 70% but took up <20% of MeHg, while P. mercurii ND132 adsorbed <20% but took up negligible amounts of MeHg. These results suggest that microbial surface adsorption and intracellular uptake of Hg(II) and MeHg depend on the specific types of microbes and appear to be related to microbial physiology that requires further detailed investigation. Despite being incapable of methylating Hg(II), methanotrophs play important roles in immobilizing both Hg(II) and MeHg, potentially influencing their bioavailability and trophic transfer. Therefore, methanotrophs are not only important sinks for methane but also for Hg(II) and MeHg and can influence the global cycling of C and Hg.
Topics: Methylmercury Compounds; Mercury; Adsorption; Methylation; Bacteria
PubMed: 37187279
DOI: 10.1016/j.envpol.2023.121790 -
BMC Musculoskeletal Disorders Aug 2022Congenital scoliosis (CS) is a congenital deformity of the spine resulting from abnormal and asymmetrical development of vertebral bodies during pregnancy. However, the...
BACKGROUND
Congenital scoliosis (CS) is a congenital deformity of the spine resulting from abnormal and asymmetrical development of vertebral bodies during pregnancy. However, the etiology and mechanism of CS remain unclear. Epigenetics is the study of heritable variations in gene expression outside of changes in nucleotide sequence. Among these, DNA methylation was described first and is the most characteristic and most stable epigenetic mechanism. Therefore, in this study, we aim to explore the association between genome methylation and CS which are not been studied before.
METHODS
Two pairs of monozygotic twins were included, with each pair involving one individual with and one without CS. Agilent SureSelect XT Human Methyl-Sequencing was used for genome methylation sequencing. MethylTarget was used to detect methylation levels in target regions. Immunohistochemistry was performed to visualize expression of associated genes in candidate regions.
RESULTS
A total of 75 differentially methylated regions were identified, including 24 with an increased methylation level and 51 with a decreased methylation level in the CS group. Nine of the differentially methylated regions were selected (TNS3, SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, and GRHL2). The results showed that the methylation level of the promoter region of TNS3 was 0.72 ± 0.08 in the CS group and 0.43 ± 0.06 in the control group (p = 0.00070 < 0.01). There was no significant difference in the degree of methylation of SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, or GRHL2 between the two groups. Immunohistochemistry showed significantly decreased TNS3 expression in the cartilage of the articular process in CS (CS: 0.011 ± 0.002; control: 0.018 ± 0.006, P = 0.003 < 0.01).
CONCLUSION
Compared with the control group, high-level methylation of the TNS3 promoter region and low TNS3 expression in the cartilage layer of the articular process characterize CS. Thus, DNA methylation and TNS3 may play important roles in the pathogenesis of CS.
Topics: Base Sequence; DNA Methylation; Epigenesis, Genetic; Female; Humans; Pregnancy; Scoliosis; Tensins
PubMed: 35987623
DOI: 10.1186/s12891-022-05730-x -
Pharmacology & Therapeutics Jul 2023Tumor endothelial cells (TECs) reside in the inner lining of blood vessels and represent a promising target for targeted cancer therapy. DNA methylation is a chemical... (Review)
Review
Tumor endothelial cells (TECs) reside in the inner lining of blood vessels and represent a promising target for targeted cancer therapy. DNA methylation is a chemical process that involves the transfer of a methyl group to a specific base in the DNA strand, catalyzed by a DNA methyltransferase (DNMT). DNMT inhibitors (DNMTis) can inhibit the activity of DNMTs, thereby preventing the transfer of methyl groups from s-adenosyl methionine (SAM) to cytosine. Currently, the most viable therapy for TECs is the development of DNMTis to release cancer suppressor genes from their repressed state. In this review, we first outline the characteristics of TECs and describe the development of tumor blood vessels and TECs. Abnormal DNA methylation is closely linked to tumor initiation, progression, and cell carcinogenesis, as evidenced by numerous studies. Therefore, we summarize the role of DNA methylation and DNA methyltransferase and the therapeutic potential of four types of DNMTi in targeting TECs. Finally, we discuss the accomplishments, challenges, and opportunities associated with combination therapy with DNMTis for TECs.
Topics: Humans; Endothelial Cells; Neoplasms; DNA Methylation; Methyltransferases; DNA Modification Methylases; DNA; Enzyme Inhibitors
PubMed: 37172786
DOI: 10.1016/j.pharmthera.2023.108434 -
Journal of the History of Biology Dec 2022DNA methylation is a quintessential epigenetic mechanism. Widely considered a stable regulator of gene silencing, it represents a form of "molecular braille," chemically... (Review)
Review
DNA methylation is a quintessential epigenetic mechanism. Widely considered a stable regulator of gene silencing, it represents a form of "molecular braille," chemically printed on DNA to regulate its structure and the expression of genetic information. However, there was a time when methyl groups simply existed in cells, mysteriously speckled across the cytosine building blocks of DNA. Why was the code of life chemically modified, apparently by "no accident of enzyme action" (Wyatt 1951)? If all cells in a body share the same genome sequence, how do they adopt unique functions and maintain stable developmental states? Do cells remember? In this historical perspective, I review epigenetic history and principles and the tools, key scientists, and concepts that brought us the synthesis and discovery of prokaryotic and eukaryotic methylated DNA. Drawing heavily on Gerard Wyatt's observation of asymmetric levels of methylated DNA across species, as well as to a pair of visionary 1975 DNA methylation papers, 5-methylcytosine is connected to DNA methylating enzymes in bacteria, the maintenance of stable cellular states over development, and to the regulation of gene expression through protein-DNA binding. These works have not only shaped our views on heritability and gene regulation but also remind us that core epigenetic concepts emerged from the intrinsic requirement for epigenetic mechanisms to exist. Driven by observations across prokaryotic and eukaryotic worlds, epigenetic systems function to access and interpret genetic information across all forms of life. Collectively, these works offer many guiding principles for our epigenetic understanding for today, and for the next generation of epigenetic inquiry in a postgenomics world.
Topics: DNA Methylation; Epigenesis, Genetic; DNA; Gene Silencing; Eukaryota; Writing
PubMed: 36239862
DOI: 10.1007/s10739-022-09691-8