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Applied Microbiology and Biotechnology Feb 2024Methylmercury formation is mainly driven by microbial-mediated process. The mechanism of microbial mercury methylation has become a crucial research topic for... (Review)
Review
Methylmercury formation is mainly driven by microbial-mediated process. The mechanism of microbial mercury methylation has become a crucial research topic for understanding methylation in the environment. Pioneering studies of microbial mercury methylation are focusing on functional strain isolation, microbial community composition characterization, and mechanism elucidation in various environments. Therefore, the functional genes of microbial mercury methylation, global isolations of Hg methylation strains, and their methylation potential were systematically analyzed, and methylators in typical environments were extensively reviewed. The main drivers (key physicochemical factors and microbiota) of microbial mercury methylation were summarized and discussed. Though significant progress on the mechanism of the Hg microbial methylation has been explored in recent decade, it is still limited in several aspects, including (1) molecular biology techniques for identifying methylators; (2) characterization methods for mercury methylation potential; and (3) complex environmental properties (environmental factors, complex communities, etc.). Accordingly, strategies for studying the Hg microbial methylation mechanism were proposed. These strategies include the following: (1) the development of new molecular biology methods to characterize methylation potential; (2) treating the environment as a micro-ecosystem and studying them from a holistic perspective to clearly understand mercury methylation; (3) a more reasonable and sensitive inhibition test needs to be considered. KEY POINTS: • Global Hg microbial methylation is phylogenetically and functionally discussed. • The main drivers of microbial methylation are compared in various condition. • Future study of Hg microbial methylation is proposed.
Topics: Mercury; Microbiota; Protein Processing, Post-Translational; Methylation
PubMed: 38407657
DOI: 10.1007/s00253-023-12967-6 -
Methods in Enzymology 2023Protein α-N-terminal (Nα) methylation is a post-translational modification catalyzed by N-terminal methyltransferase 1/2 (NTMT1/2) and METTL13. Nα methylation affects...
Protein α-N-terminal (Nα) methylation is a post-translational modification catalyzed by N-terminal methyltransferase 1/2 (NTMT1/2) and METTL13. Nα methylation affects protein stability, protein-protein interaction, and protein-DNA interaction. Thus, Nα methylated peptides are essential tools to study the function of Nα methylation, generate specific antibodies for different states of Nα methylation, and characterize the enzyme kinetics and activity. Here, we describe chemical methods of site-specific synthesis of Nα mono-, di-, and trimethylated peptides in the solid phase. In addition, we describethe preparation of trimethylation peptides by recombinant NTMT1 catalysis.
Topics: Methylation; Peptides; Protein Processing, Post-Translational
PubMed: 37230586
DOI: 10.1016/bs.mie.2023.02.008 -
Genome Biology and Evolution Apr 2023The origin of microbial mercury methylation has long been a mystery. Here, we employed genome-resolved phylogenetic analyses to decipher the evolution of the...
The origin of microbial mercury methylation has long been a mystery. Here, we employed genome-resolved phylogenetic analyses to decipher the evolution of the mercury-methylating gene, hgcAB, constrain the ancestral origin of the hgc operon, and explain the distribution of hgc in Bacteria and Archaea. We infer the extent to which vertical inheritance and horizontal gene transfer have influenced the evolution of mercury methylators and hypothesize that evolution of this trait bestowed the ability to produce an antimicrobial compound (MeHg+) on a potentially resource-limited early Earth. We speculate that, in response, the evolution of MeHg+-detoxifying alkylmercury lyase (encoded by merB) reduced a selective advantage for mercury methylators and resulted in widespread loss of hgc in Bacteria and Archaea.
Topics: Mercury; Methylmercury Compounds; Methylation; Phylogeny; Bacteria; Archaea
PubMed: 36951100
DOI: 10.1093/gbe/evad051 -
The Journal of Biological Chemistry Jul 2021Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the...
Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2-Rpb1-interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.
Topics: Animals; COS Cells; Catalytic Domain; Chlorocebus aethiops; Histone-Lysine N-Methyltransferase; Histones; Humans; Methylation; Mutation; Protein Binding; Protein Processing, Post-Translational; Tubulin
PubMed: 34157286
DOI: 10.1016/j.jbc.2021.100898 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
Frontiers in Endocrinology 2022CpG island methylator phenotype (CIMP), characterized by the concurrent and widespread hypermethylation of a cluster of CpGs, has been reported to play an important role...
CpG island methylator phenotype (CIMP), characterized by the concurrent and widespread hypermethylation of a cluster of CpGs, has been reported to play an important role in carcinogenesis. Limited studies have explored the role of CIMP in papillary thyroid carcinomas (PTCs). Here, in genome-wide DNA methylation analysis of 350 primary PTCs from the Cancer Genome Atlas database that were assessed using the Illumina HumanMethylation450K platform, our study helps to identify two subtypes displayed markedly distinct DNA methylation levels, termed CIMP (high levels of DNA methylation) and nCIMP subgroup (low levels of DNA methylation). Interestingly, PTCs with CIMP tend to have a higher degree of malignancy, since this subtype was tightly associated with older age, advanced pathological stage, and lymph node metastasis (all < 0.05). Differential methylation analysis showed a broad methylation gain in CIMP and subsequent generalized gene set testing analysis based on the significantly methylated probes in CIMP showed remarkable enrichment in epithelial mesenchymal transition and angiogenesis hallmark pathways, confirming that the CIMP phenotype may promote the tumor progression from another perspective. Analysis of tumor microenvironment showed that CIMP PTCs are in an immune-depletion status, which may affect the effectiveness of immunotherapy. Genetically, the significantly higher tumor mutation burden and copy number alteration both at the genome and focal level confirmed the genomic heterogeneity and chromosomal instability of CIMP. tumor Corresponding to the above findings, PTC patients with CIMP showed remarkable poor clinical outcome as compared to nCIMP regarding overall survival and progression-free survival. More importantly, CIMP was associated with worse survival independent of known prognostic factors.
Topics: Humans; CpG Islands; Thyroid Cancer, Papillary; DNA Methylation; Phenotype; Thyroid Neoplasms; Tumor Microenvironment
PubMed: 36353231
DOI: 10.3389/fendo.2022.1008301 -
Journal of Translational Medicine Feb 2021Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the...
BACKGROUND
Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC).
METHODS
An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP).
RESULTS
The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma.
CONCLUSIONS
This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.
Topics: Biomarkers, Tumor; Colorectal Neoplasms; DNA Methylation; Humans; Prognosis; Receptor, trkC; Retrospective Studies; Tropomyosin
PubMed: 33593392
DOI: 10.1186/s12967-021-02740-6 -
Bioinformatics (Oxford, England) Sep 2019The identification of differentially methylated regions (DMRs) among phenotypes is one of the main goals of epigenetic analysis. Although there are several methods...
MOTIVATION
The identification of differentially methylated regions (DMRs) among phenotypes is one of the main goals of epigenetic analysis. Although there are several methods developed to detect DMRs, most of them are focused on detecting relatively large differences in methylation levels and fail to detect moderate, but consistent, methylation changes that might be associated to complex disorders.
RESULTS
We present mCSEA, an R package that implements a Gene Set Enrichment Analysis method to identify DMRs from Illumina450K and EPIC array data. It is especially useful for detecting subtle, but consistent, methylation differences in complex phenotypes. mCSEA also implements functions to integrate gene expression data and to detect genes with significant correlations among methylation and gene expression patterns. Using simulated datasets we show that mCSEA outperforms other tools in detecting DMRs. In addition, we applied mCSEA to a previously published dataset of sibling pairs discordant for intrauterine hyperglycemia exposure. We found several differentially methylated promoters in genes related to metabolic disorders like obesity and diabetes, demonstrating the potential of mCSEA to identify DMRs not detected by other methods.
AVAILABILITY AND IMPLEMENTATION
mCSEA is freely available from the Bioconductor repository.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: CpG Islands; DNA Methylation; Promoter Regions, Genetic; Software
PubMed: 30753302
DOI: 10.1093/bioinformatics/btz096 -
Epigenetics & Chromatin Oct 20205' methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and...
BACKGROUND
5' methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.
RESULTS
Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions.
CONCLUSION
We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.
Topics: Arabidopsis; DNA Methylation; Epigenome; Genome, Plant; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 33028374
DOI: 10.1186/s13072-020-00361-9 -
Trends in Biochemical Sciences May 2013Methylated lysine and arginine residues in histones represent a crucial part of the histone code, and recognition of these methylated residues by protein interaction... (Review)
Review
Methylated lysine and arginine residues in histones represent a crucial part of the histone code, and recognition of these methylated residues by protein interaction domains modulates transcription. Although some methylating enzymes appear to be histone specific, many can modify histone and non-histone substrates and an increasing number are specific for non-histone substrates. Some of the non-histone substrates can also be involved in transcription, but a distinct subset of protein methylation reactions occurs at residues buried deeply in ribosomal proteins that may function in protein-RNA interactions rather than protein-protein interactions. Additionally, recent work has identified enzymes that catalyze protein methylation reactions at new sites in ribosomal and other proteins. These reactions include modifications of histidine and cysteine residues as well as the N terminus.
Topics: Animals; Histone-Lysine N-Methyltransferase; Histones; Humans; Methylation; Protein Interaction Domains and Motifs; Proteins; Ribosomal Proteins; Transcription Factors
PubMed: 23490039
DOI: 10.1016/j.tibs.2013.02.004