Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.

Metabolic engineering of Corynebacterium glutamicum ATCC13032 to produce S-adenosyl-L-methionine.

As an important biological methyl group donor, S-adenosyl-L-methionine is used as nutritional supplement or drug for various diseases, but bacterial strains that can efficiently produce S-adenosyl-L-methionine are not available. In this study, Corynebacterium glutamicum strain HW104 which can accumulate S-adenosyl-L-methionine was constructed from C. glutamicum ATCC13032 by deleting four genes thrB, metB, mcbR and Ncgl2640, and six genes metK, vgb, lysC(m), hom(m), metX and metY were overexpressed in HW104 in different combinations, forming strains HW104/pJYW-4-metK-vgb, HW104/pJYW-4-SAM2C-vgb, HW104/pJYW-4-metK-vgb-metYX, and HW104/pJYW-4-metK-vgb-metYX-hom(m)-lysC(m). Fermentation experiments showed that HW104/pJYW-4-metK-vgb produced more S-adenosyl-L-methionine than other strains, and the yield achieved 196.7 mg/L (12.15 mg/g DCW) after 48h. The results demonstrate the potential application of C. glutamicum for production of S-adenosyl-L-methionine without addition of L-methionine.

Regulation of Folate-Mediated One-Carbon Metabolism by Glycine N-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR).

Folate-mediated one-carbon metabolism is an important therapeutic target of human diseases. We extensively investigated how gene-nutrient interactions may modulate human cancer risk in 2 major folate metabolic genes, MTHFR and GNMT. The biochemical impacts of MTHFR and GNMT on methyl group supply, global DNA methylation, nucleotide biosynthesis, DNA damage, and partitioning of the folate dependent 1-carbon group were carefully studied. The distinct model systems used included: EB virus-transformed lymphoblasts expressing human MTHFR polymorphic genotypes; liver-derived GNMT-null cell-lines with and without GNMT overexpression; and HepG2 cells with stabilized inhibition of MTHFR using shRNA, GNMT wildtype, heterozygotous (GNMT(het)) and knockout (GNMT(nul)) mice. We discovered that the MTHFR TT genotype significantly reduces folate-dependent remethylation under folate restriction, but it assists purine synthesis when folate is adequate. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in human hematological malignancies. GNMT affects transmethylation kinetics and S-adenosylmethionine (adoMet) synthesis, and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. Restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. The systematic series of studies gives new insights into the underlying mechanisms by which MTHFR and GNMT may participate in human tumor prevention.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

Co-production of S-adenosyl-L-methionine and L-isoleucine in Corynebacterium glutamicum.

In this study, production of S-adenosyl-L-methionine in Corynebacterium glutamicum was investigated by overexpressing genes metK and vgb. Compared with vector control, overexpression of metK alone in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.11 and 11.65 times, respectively; while overexpression of metK and vgb in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.83 and 14.95 times, respectively. Further studies on IWJ001/pDXW-8-metk-vgb showed that the limiting factor for SAM production is intracellular ATP supply. Since IWJ001 is an L-isoleucine production strain, IWJ001/pDXW-8-metk-vgb could produce both SAM and L-isoleucine. After 72 h fermentation, SAM and L-isoleucine in IWJ001/pDXW-8-metk-vgb reached 0.67 g/L and 13.8 g/L, respectively. The results demonstrate the potential application of C. glutamicum for co-production of SAM and amino acids.

Control of ATP concentration in Escherichia coli using synthetic small regulatory RNAs for enhanced S-adenosylmethionine production.

ATP is the limiting precursor and driving force for S-adenosylmethionine (SAM) biosynthesis in Escherichia coli. In contrast to traditional optimization of fermentation processes, the synthetic sRNA-based repression strategy, which was developed as a highly efficient gene knockdown approach, has been applied for the regulation of the intracellular ATP concentration in order to enhance SAM production. In this work, proB, glnA and argB, all involved in the synthesis of ATP-dependent by-products in the S-adenosylmethionine production were selected as candidates for repression. The results show that the S-adenosylmethionine titer and yield in the recombinant strain were doubled compared with the control. The best-performing strain, Anti-argB, produced the highest SAM titer (1.21 mg L(-1)), and strain Anti-glnA gave the highest yield (0.13 mg g(-1), 12 h). Both the concentration of ATP and the ratio of ATP to ADP were shown to have a positive effect on the S-adenosylmethionine synthesis. Overall, the synthetic sRNA-based downregulation strategy has a high potential for cofactor regulation and will be useful for industrial ATP-driven bioprocesses.

A thermostable archaeal S-adenosylmethionine synthetase: a promising tool to improve the synthesis of adenosylmethionine analogs of biotechnological interest.

The naturally and widely occurring sulfonium compound, S-adenosylmethionine (AdoMet), one of nature's most versatile molecules, is biosynthesized from methionine and ATP by AdoMet synthetase or methionine adenosyltransferase (MAT) in a 2-step reaction in which the energy-rich sulfonium compound is formed by dephosphorylation of ATP. All living cells, with the only exception of some parasites and infectious agents, express MAT.

(13)C-metabolic flux analysis in S-adenosyl-L-methionine production by Saccharomyces cerevisiae.

S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

Selection and characterization of Cheonggukjang (fast fermented soybean paste)-originated bacterial strains with a high level of S-adenosyl-L-methionine production and probiotics efficacy.

This study was executed to develop probiotics producing S-adenosyl-L-methionine (SAMe), a methyl group donor in the 5-methyltetrahydrofolate methylation reaction in animal cells. SAMe is an essential substance in the synthesis, activation, and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and cell membranes of animals. SAMe is also known as a nutritional supplement for improving human brain function. In this study, SAMe-producing strains were identified in six kinds of Cheonggukjang, and strains with excellent SAMe production were identified, with one strain in the Enterococcus genus and six strains in the Bacillus genus. Strains with a large amount of SAMe production included lactic acid bacteria, such as Enterococcus faecium, Enterococcus durans, and Enterococcus sanguinicola, as well as various strains in the Bacillus genus. The SAMe-overproducing strains showed antibacterial activity against some harmful microbes, in addition to weak acid resistance and strong bile resistance, indicating characteristics of probiotics. Cheonggukjang-originated beneficial bacterial strains overproducing SAMe may be commercially useful for manufacturing SAMe-rich foods.

Enhancing precursors availability in Pichia pastoris for the overproduction of S-adenosyl-L-methionine employing molecular strategies with process tuning.

S-Adenosyl-L-methionine (SAM) is an important metabolite having prominent role in treating various diseases. Due to increasing demand of SAM, improvement in its production is essential. For this purpose, S-adenosyl-l-methionine synthetase gene (sam2) was overexpressed in the present study, and we studied the effect of coexpression of methionine permease (mup1) and adenylate kinase (adk1) genes. From the recombinant strains expressing individual genes, we observed that SAM2 synthetase is the primary limiting factor and its overexpression is essential to increase the SAM productivity. Coexpression of mup1 with sam2 did not enhance SAM production, while coexpression of adk1 with sam2 improved SAM production, clearly indicating that ATP is the primary limiting precursor in SAM production. However, coexpression of all three genes synergistically improved SAM productivity with better L-methionine (L-met) conversion efficiency in every stage, and it was 77% more compared to overexpressing sam2 alone. Sparging pure oxygen reduced cultivation time. Feeding nitrogen source and additional L-met during induction phase enhanced SAM yield by 38.4% and 55.1%, respectively. Moreover, building up biomass before induction resulted in 145% increase in specific yield and 83% higher L-met conversion efficiency. This is the first report on increasing both the precursors L-met and ATP availability through molecular strategies using microorganisms for the production of SAM.

Glycine N-methyltransferase expression in the hippocampus and its role in neurogenesis and cognitive performance.

The hippocampus is a brain area characterized by its high plasticity, observed at all levels of organization: molecular, synaptic, and cellular, the latter referring to the capacity of neural precursors within the hippocampus to give rise to new neurons throughout life. Recent findings suggest that promoter methylation is a plastic process subjected to regulation, and this plasticity seems to be particularly important for hippocampal neurogenesis. We have detected the enzyme GNMT (a liver metabolic enzyme) in the hippocampus. GNMT regulates intracellular levels of SAMe, which is a universal methyl donor implied in almost all methylation reactions and, thus, of prime importance for DNA methylation. In addition, we show that deficiency of this enzyme in mice (Gnmt-/-) results in high SAMe levels within the hippocampus, reduced neurogenic capacity, and spatial learning and memory impairment. In vitro, SAMe inhibited neural precursor cell division in a concentration-dependent manner, but only when proliferation signals were triggered by bFGF. Indeed, SAMe inhibited the bFGF-stimulated MAP kinase signaling cascade, resulting in decreased cyclin E expression. These results suggest that alterations in the concentration of SAMe impair neurogenesis and contribute to cognitive decline.

2,4-Dichlorophenol induces global DNA hypermethylation through the increase of S-adenosylmethionine and the upregulation of DNMTs mRNA in the liver of goldfish Carassius auratus.

Altered DNA methylation is associated with changes in gene expression, signal transduction and stress response after exposure to a wide range of exogenous compounds, and abnormal methylation is a major toxic effect induced by chemicals such as benzene and phenols. 2,4-Dichlorophenol (2,4-DCP), a derivative of phenol, has been classified as a priority pollutant by the US EPA due to its toxic effects on aquatic organisms. However, the effect of 2,4-DCP on DNA methylation and its potential mechanism in fish are rarely understood. The present study aims to figure out whether 2,4-DCP could impact DNA methylation and explore its potential mechanisms by measuring the global DNA methylation levels, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) contents, the mRNA expression of DNA methyltransferase1 (DNMT1) and DNA methyltransferase3 (DNMT3) in the liver of goldfish Carassius auratus. DNA methylation levels were analyzed using high performance liquid chromatography (HPLC) and MspI/HpaII ethidium bromide assay, SAM and SAH contents were determined by HPLC, the mRNA expression of DNMT1 and DNMT3 was measured by quantitative-PCR (qPCR). The results showed that 2,4-DCP caused global DNA hypermethylation, elevated the methylation levels of CpG islands, increased the SAM and SAH contents, decreased the SAM/SAH ratio, and upregulated the mRNA expression of DNMT1 and DNMT3, while depletion of SAM with Na2SeO3 and inhibition of DNMTs activity with 5-aza-2'-deoxycytidine (5AdC) impaired 2,4-DCP-induced global DNA hypermethylation, suggesting that the increase of SAM contents and upregulation of the mRNA expression of DNMT1 and DNMT3 may play important roles in 2,4-DCP-induced global DNA hypermethylation process. Our report is the first one to show that short-term 2,4-DCP exposure caused the global DNA hypermethylation via altered SAM level and DNMTs expression in fish.

Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs.

Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalysed modification of biomolecules including proteins, nucleic acids, and small molecules. However, SAM and its analogs suffer from an inherent instability, and their chemical synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymatic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Molecular modelling studies are used to guide the rational design of mutants, and the enzymatic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymatic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chemical synthesis and can be applied in situ to overcome stability and activity issues.

Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function.

The identification of novel metabolites and the characterization of their biological functions are major challenges in biology. X-ray crystallography can reveal unanticipated ligands that persist through purification and crystallization. These adventitious protein-ligand complexes provide insights into new activities, pathways and regulatory mechanisms. We describe a new metabolite, carboxy-S-adenosyl-l-methionine (Cx-SAM), its biosynthetic pathway and its role in transfer RNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand consistent with Cx-SAM in the catalytic site. Mechanistic analyses showed an unprecedented role for prephenate as the carboxyl donor and the involvement of a unique ylide intermediate as the carboxyl acceptor in the CmoA-mediated conversion of SAM to Cx-SAM. A second member of the SAM-dependent methyltransferase superfamily, CmoB, recognizes Cx-SAM and acts as a carboxymethyltransferase to convert 5-hydroxyuridine into 5-oxyacetyl uridine at the wobble position of multiple tRNAs in Gram-negative bacteria, resulting in expanded codon-recognition properties. CmoA and CmoB represent the first documented synthase and transferase for Cx-SAM. These findings reveal new functional diversity in the SAM-dependent methyltransferase superfamily and expand the metabolic and biological contributions of SAM-based biochemistry. These discoveries highlight the value of structural genomics approaches in identifying ligands within the context of their physiologically relevant macromolecular binding partners, and in revealing their functions.

Inhibition of proteolysis in histiotrophic nutrition pathways alters DNA methylation and one-carbon metabolism in the organogenesis-stage rat conceptus.

Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM), is biosynthesized from methionine and adenosine triphosphate by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 µM leupeptin in whole embryo culture for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. After 6 h on GD11, the 100-µM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels and inhibited Mat2a specific activity, all of which produced a significant 52% reduction of SAM in the VYS. The 50- and 100-µM leupeptin treatments significantly decreased global methylation levels by 6%-9% in EMB and by 11%-15% in VYS following both 6- and 26-h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation.

Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits.

MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.

Improved co-production of S-adenosylmethionine and glutathione using citrate as an auxiliary energy substrate.

The effects of sodium citrate on the fermentative co-production of S-adenosylmethionine (SAM) and glutathione (GSH) using Candida utilis CCTCC M 209298 were investigated. Sodium citrate was beneficial for the biosynthesis of SAM and GSH and in turn improved intracellular SAM and GSH contents. Adding 2 g/L of sodium citrate at 15 h was the most efficient approach for achieving elevated co-production of SAM and GSH. Using this sodium citrate addition mode, co-production of SAM and GSH reached 663.9 mg/L, which was increased by 27.5% compared to the control. Based on analysis of the kinetic parameters, evaluation of the energy metabolism and assay of key enzymes, sodium citrate was verified to act as an auxiliary energy substrate for the overproduction of SAM and GSH.

Cross-talk between sulfur assimilation and ethylene signaling in plants.

Sulfur (S) deficiency is prevailing all over the world and becoming an important issue for crop improvement through maximising its utilization efficiency by plants for sustainable agriculture. Its interaction with other regulatory molecules in plants is necessary to improve our understanding on its role under changing environment. Our knowledge on the influence of S on ethylene signaling is meagre although it is a constituent of cysteine (Cys) required for the synthesis of reduced glutathione (GSH) and S-adenosyl methionine (SAM), a precursor of ethylene biosynthesis. Thus, there may be an interaction between S assimilation, ethylene signaling and plant responses under optimal and stressful environmental conditions. The present review emphasizes that responses of plants to S involve ethylene action. This evaluation will provide an insight into the details of interactive role of S and ethylene signaling in regulating plant processes and prove profitable for developing sustainability under changing environmental conditions.

Metabolic aspects of epigenome: coupling of S-adenosylmethionine synthesis and gene regulation on chromatin by SAMIT module.

Histone and DNA methyltransferases utilize S-adenosyl-L-methionine (SAM), a key intermediate of sulfur amino acid metabolism, as a donor of methyl group. SAM is biosynthesized by methionine adenosyltransferase (MAT) using two substrates, methionine and ATP. Three distinct forms of MAT (MATI, MATII and MATIII), encoded by two distinct genes (MAT1A and MAT2A), have been identified in mammals. MATII consists of α2 catalytic subunit encoded by MAT2A and ß regulatory subunit encoded by MAT2B, but the physiological function of the ß subunit is not clear. MafK is a member of Maf oncoproteins and functions as both transcription activator and repressor by forming diverse heterodimers to bind to DNA elements termed Maf recognition elements. Proteomics analysis of MafK-interactome revealed its interaction with both MATIIα and MATIIß. They are recruited specifically to MafK target genes and are required for their repression by MafK and its partner Bach1. Because the catalytic activity of MATIIα is required for the MafK target gene repression, MATIIα is suggested to provide SAM locally on chromatin where it is recruited. One of the unexpected features of MATII is that MATIIα interacts with many chromatin-related proteins of diverse functions such as histone modification, chromatin remodeling, transcription regulation, and nucleo-cytoplasmic transport. MATIIα appears to generate multiple, heterogenous regulatory complexes where it provides SAM. Considering their function, the heterooligomer of MATIIα and ß is named SAMIT (SAM-integrating transcription) module within their interactome where it serves SAM for nuclear methyltransferases.

Profiling genome-wide chromatin methylation with engineered posttranslation apparatus within living cells.

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.