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Gene Expression 2000DNA methylation is a major determinant in the epigenetic silencing of genes. The mechanisms underlying the targeting of DNA methylation and the subsequent repression of... (Review)
Review
DNA methylation is a major determinant in the epigenetic silencing of genes. The mechanisms underlying the targeting of DNA methylation and the subsequent repression of transcription are relevant to human development and disease, as well as for attempts at somatic gene therapy. The success of transgenic technologies in plants and animals is also compromised by DNA methylation-dependent silencing pathways. Recent biochemical experiments provide a mechanistic foundation for understanding the influence of DNA methylation on transcription. The DNA methyltransferase Dnmt1, and several methyl-CpG binding proteins, MeCP2, MBD2, and MBD3, all associate with histone deacetylase. These observations firmly connect DNA methylation with chromatin modifications. They also provide new pathways for the potential targeting of DNA methylation to repressive chromatin as well as the assembly of repressive chromatin on methylated DNA. Here we discuss the implications of the methylation-acetylation connection for human cancers and the developmental syndromes Fragile X and Rett, which involve a mistargeting of DNA methylation-dependent repression.
Topics: Animals; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation; Gene Expression Regulation, Developmental; Genetic Diseases, Inborn; Histone Deacetylases; Histones; Humans
PubMed: 11097425
DOI: 10.3727/000000001783992731 -
The ISME Journal Jun 2021Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent neurotoxin that accumulates in terrestrial and marine food webs, with potential impacts on...
Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent neurotoxin that accumulates in terrestrial and marine food webs, with potential impacts on human health. This process requires the gene pair hgcAB, which encodes for proteins that actuate Hg methylation, and has been well described for anoxic environments. However, recent studies report potential MeHg formation in suboxic seawater, although the microorganisms involved remain poorly understood. In this study, we conducted large-scale multi-omic analyses to search for putative microbial Hg methylators along defined redox gradients in Saanich Inlet, British Columbia, a model natural ecosystem with previously measured Hg and MeHg concentration profiles. Analysis of gene expression profiles along the redoxcline identified several putative Hg methylating microbial groups, including Calditrichaeota, SAR324 and Marinimicrobia, with the last the most active based on hgc transcription levels. Marinimicrobia hgc genes were identified from multiple publicly available marine metagenomes, consistent with a potential key role in marine Hg methylation. Computational homology modelling predicts that Marinimicrobia HgcAB proteins contain the highly conserved amino acid sites and folding structures required for functional Hg methylation. Furthermore, a number of terminal oxidases from aerobic respiratory chains were associated with several putative novel Hg methylators. Our findings thus reveal potential novel marine Hg-methylating microorganisms with a greater oxygen tolerance and broader habitat range than previously recognized.
Topics: Bacteria; British Columbia; Ecosystem; Humans; Mercury; Methylation; Water Pollutants, Chemical
PubMed: 33504941
DOI: 10.1038/s41396-020-00889-4 -
Biological Chemistry Aug 2013Asymmetric dimethylation of arginine side chains in proteins is a frequent posttranslational modification, catalyzed by type I protein arginine methyltransferases... (Review)
Review
Asymmetric dimethylation of arginine side chains in proteins is a frequent posttranslational modification, catalyzed by type I protein arginine methyltransferases (PRMTs). This article summarizes what is known about this modification in the nuclear poly(A)-binding protein (PABPN1). PABPN1 contains 13 dimethylated arginine residues in its C-terminal domain. Three enzymes, PRMT1, 3, and 6, can methylate PABPN1. Although 26 methyl groups are transferred to one PABPN1 molecule, the PRMTs do so in a distributive reaction, i.e., only a single methyl group is transferred per binding event. As PRMTs form dimers, with the active sites accessible from a small central cavity, backbone conformation around the methyl-accepting arginine is an important determinant of substrate specificity. Neither the association of PABPN1 with poly(A) nor its role in poly(A) tail synthesis is affected by arginine methylation. At least at low protein concentration, methylation does not affect the protein's tendency to oligomerize. The dimethylarginine residues of PABPN1 are located in the binding site for its nuclear import receptor, transportin. Arginine methylation weakens this interaction about 10-fold. Very recent evidence suggests that arginine methylation as a way of fine-tuning the interactions between transportin and its cargo may be a general mechanism.
Topics: Animals; Humans; Methylation; Models, Molecular; Poly(A)-Binding Protein I; Protein Conformation; Protein-Arginine N-Methyltransferases; Substrate Specificity
PubMed: 23412876
DOI: 10.1515/hsz-2013-0121 -
Proceedings of the National Academy of... Jun 2023Defining all sites for a post-translational modification in the cell, and identifying their upstream modifying enzymes, is essential for a complete understanding of a...
Defining all sites for a post-translational modification in the cell, and identifying their upstream modifying enzymes, is essential for a complete understanding of a modification's function. However, the complete mapping of a modification in the proteome and definition of its associated enzyme-substrate network is rarely achieved. Here, we present the protein methylation network for Through a formal process of defining and quantifying all potential sources of incompleteness, for both the methylation sites in the proteome and also protein methyltransferases, we prove that this protein methylation network is now near-complete. It contains 33 methylated proteins and 28 methyltransferases, comprising 44 enzyme-substrate relationships, and a predicted further three enzymes. While the precise molecular function of most methylation sites is unknown, and it remains possible that other sites and enzymes remain undiscovered, the completeness of this protein modification network is unprecedented and allows us to holistically explore the role and evolution of protein methylation in the eukaryotic cell. We show that while no single protein methylation event is essential in yeast, the vast majority of methylated proteins are themselves essential, being primarily involved in the core cellular processes of transcription, RNA processing, and translation. This suggests that protein methylation in lower eukaryotes exists to fine-tune proteins whose sequences are evolutionarily constrained, providing an improvement in the efficiency of their cognate processes. The approach described here, for the construction and evaluation of post-translational modification networks and their constituent enzymes and substrates, defines a formal process of utility for other post-translational modifications.
Topics: Saccharomyces cerevisiae; Methylation; Saccharomyces cerevisiae Proteins; Eukaryotic Cells; Proteome; Protein Processing, Post-Translational
PubMed: 37252976
DOI: 10.1073/pnas.2215431120 -
Journal of Advanced Research Dec 2022Histone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure...
INTRODUCTION
Histone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure and gene expression. However, the involvement of non-histone methylation in plant biological processes remains largely unknown.
METHODS
The methylated substrates and methylation sites during tomato fruit ripening were identified by LC-MS/MS. Bioinformatics of lysine methylated proteins was conducted to analyze the possible role of methylated proteins. The effects of methylation modification on protein functions were preliminarily investigated by site-directed mutation simulation.
RESULTS
A total of 241 lysine methylation (mono-, di- and trimethylation) sites in 176 proteins were identified with two conserved methylation motifs: xxxxxxExxx_K_xxxExxxxxx and xxxxxxExxx_K_xxxxxxxxxx. These methylated proteins were mainly related to fruit ripening and senescence, oxidation reduction process, signal transduction, stimulus and stress responses, and energy metabolism. Three representative proteins, thioredoxin (Trx), glutathione S-transferase T1 (GST T1), and NADH dehydrogenase (NOX), were selected to investigate the effect of methylation modifications on protein activity. Mimicking demethylation led to decreased Trx activity but increased GST T1 and NOX activities. In addition, RT-qPCR exhibited that the expression of many genes that encode proteins subjected to methylation was upregulated during fruit ripening.
CONCLUSION
Our study suggests that tomato fruit ripening undergo non-histone lysine methylation, which may participate in the regulation of fruit ripening. It is the first report of methyl proteome profiling of non-histone lysine in horticultural crops.
Topics: Solanum lycopersicum; Methylation; Proteome; Lysine; Gene Expression Regulation, Plant; Fruit; Plant Proteins; Chromatography, Liquid; Tandem Mass Spectrometry; Histones
PubMed: 36513412
DOI: 10.1016/j.jare.2022.02.013 -
Journal of Virology Nov 2009During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently...
During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.
Topics: Amino Acid Substitution; Base Sequence; Binding Sites; DNA-Directed RNA Polymerases; Guanine; Methylation; Methyltransferases; RNA Caps; RNA, Messenger; Ribose; Vesiculovirus; Viral Proteins
PubMed: 19710136
DOI: 10.1128/JVI.01426-09 -
Transcription 2022N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions....
N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout () mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in livers and verified NRMT1 can methylate ZHX2 . In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in mice.
Topics: Animals; Cytochrome P-450 Enzyme System; Homeodomain Proteins; Methylation; Methyltransferases; Mice; Mice, Knockout; Promoter Regions, Genetic; Protein Processing, Post-Translational; Transcription Factors
PubMed: 35613330
DOI: 10.1080/21541264.2022.2079184 -
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 -
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 -
The Journal of Experimental Biology Jul 2020The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and... (Review)
Review
The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and organ-specific phenotypes from a single organismic genome. Epigenetic marks that comprise the epigenome (e.g. methylation) are placed upon or removed from chromatin (histones and DNA) to direct the activity of effectors that regulate gene expression and chromatin structure. Recently, the cytoskeleton has been identified as a second target for the cell's epigenetic machinery. Several epigenetic 'readers, writers and erasers' that remodel chromatin have been discovered to also remodel the cytoskeleton, regulating structure and function of microtubules and actin filaments. This points to an emerging paradigm for dual-function remodelers with 'chromatocytoskeletal' activity that can integrate cytoplasmic and nuclear functions. For example, the SET domain-containing 2 methyltransferase (SETD2) has chromatocytoskeletal activity, methylating both histones and microtubules. The SETD2 methyl mark on chromatin is required for efficient DNA repair, and its microtubule methyl mark is required for proper chromosome segregation during mitosis. This unexpected convergence of SETD2 activity on histones and microtubules to maintain genomic stability suggests the intriguing possibility of an expanded role in the cell for chromatocytoskeletal proteins that read, write and erase methyl marks on the cytoskeleton as well as chromatin. Coordinated use of methyl marks to remodel both the epigenome and the (epi)cytoskeleton opens the possibility for integrated regulation (which we refer to as 'epiregulation') of other higher-level functions, such as muscle contraction or learning and memory, and could even have evolutionary implications.
Topics: Chromatin; Cytoskeleton; DNA Methylation; Epigenesis, Genetic; Epigenome; Histones; Methylation; Microtubules
PubMed: 32620673
DOI: 10.1242/jeb.220632