Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-04-15

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA.

PubMed

Govindaraju, Gayathri; Jabeena, C A; Sethumadhavan, Devadathan Valiyamangalath; Rajaram, Nivethika; Rajavelu, Arumugam

2017-10-01

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

PubMed

James, Allison E; Rogovskyy, Artem S; Crowley, Michael A; Bankhead, Troy

2016-01-01

DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

Identification of a novel gene cluster participating in menaquinone (vitamin K2) biosynthesis. Cloning and sequence determination of the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene of Bacillus stearothermophilus.

PubMed

Koike-Takeshita, A; Koyama, T; Ogura, K

1997-05-09

We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396-18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of S-adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes.

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

A methyltransferase gene from arbuscular mycorrhizal fungi involved in arsenic methylation and volatilization.

PubMed

Li, Jinglong; Sun, Yuqing; Zhang, Xin; Hu, Yajun; Li, Tao; Zhang, Xuemeng; Wang, Zhi; Wu, Songlin; Wu, Zhaoxiang; Chen, Baodong

2018-06-20

Arbuscular mycorrhizal fungi (AMF), ubiquitous symbiotic fungi associated with the majority of terrestrial plants, were demonstrated to play important roles in arsenic (As) translocation and transformation in the plant-soil continuum, and substantially influence plant As tolerance. However, the direct involvement of AMF in As methylation and volatilization and their molecular mechanisms remain unsolved. Here, an arsenite methyltransferase gene RiMT-11 was identified and characterized from AM fungus Rhizophagus irregularis. Heterologous expression of RiMT-11 enhanced arsenite resistance of E. coli (Δars) through methylating As into monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and ultimately volatile trimethyl arsine (TMAs). In a two-compartment in vitro monoxenic cultivation system, methylated and volatile As were also detected from AM symbioses with arsenate addition, accompanied by strong up-regulation of RiMT-11 expression in extraradical hyphae. The present study provided direct evidence and illustrated an underlying mechanism of As methylation and volatilization by AMF, leading to a deeper insight into the role of AMF in As biogeochemical cycling. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed Central

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-01-01

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation. PMID:14705965

Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

PubMed Central

Balasubramaniyan, Natarajan; Ananthanarayanan, Meena

2012-01-01

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes. PMID:22345554

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

PubMed Central

Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

2012-01-01

Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

PubMed

Yang, Qin; He, Yijian; Kabahuma, Mercy; Chaya, Timothy; Kelly, Amy; Borrego, Eli; Bian, Yang; El Kasmi, Farid; Yang, Li; Teixeira, Paulo; Kolkman, Judith; Nelson, Rebecca; Kolomiets, Michael; L Dangl, Jeffery; Wisser, Randall; Caplan, Jeffrey; Li, Xu; Lauter, Nick; Balint-Kurti, Peter

2017-09-01

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes▿

PubMed Central

Schilhabel, Anke; Studenik, Sandra; Vödisch, Martin; Kreher, Sandra; Schlott, Bernhard; Pierik, Antonio Y.; Diekert, Gabriele

2009-01-01

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases. PMID:19011025

Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

PubMed

Byeon, Yeong; Back, Kyoungwhan

2016-08-01

Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

Obstructive heart defects associated with candidate genes, maternal obesity, and folic acid supplementation.

PubMed

Tang, Xinyu; Cleves, Mario A; Nick, Todd G; Li, Ming; MacLeod, Stewart L; Erickson, Stephen W; Li, Jingyun; Shaw, Gary M; Mosley, Bridget S; Hobbs, Charlotte A

2015-06-01

Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1,644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity. © 2015 Wiley Periodicals, Inc.

Characterization of DNA methyltransferase and demethylase genes in Fragaria vesca.

PubMed

Gu, Tingting; Ren, Shuai; Wang, Yuanhua; Han, Yuhui; Li, Yi

2016-06-01

DNA methylation is an epigenetic modification essential for gene regulations in plants, but understanding on how it is involved in fruit development, especially in non-climacteric fleshy fruit, is limited. The diploid woodland strawberry (Fragaria vesca) is an important model for non-climacteric fruit crops. In this study, we identified DNA methyltransferase genes and demethylase genes in Fragaria vesca and other angiosperm species. In accordance with previous studies, our phylogenetic analyses of those DNA methylation modifiers support the clustering of those genes into several classes. Our data indicate that whole-genome duplications and tandem duplications contributed to the expansion of those DNA methylation modifiers in angiosperms. We have further demonstrated that some DNA methylase and demethylase genes reach their highest expression levels in strawberry fleshy fruits when turning from white to red, suggesting that DNA methylation might undergo a dramatic change at the onset of fleshy fruit-ripening process. In addition, we have observed that expression of some DNA demethylase genes increases in response to various abiotic stresses including heat, cold, drought and salinity. Collectively, our study indicates a regulatory role of DNA methylation in the turning stage of non-climacteric fleshy fruit and responses to environment stimuli, and would facilitate functional studies of DNA methylation in the growth and development of non-climacteric fruits.

Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity

PubMed Central

Jacques-Fricke, Bridget T.; Gammill, Laura S.

2014-01-01

Neural crest precursors express genes that cause them to become migratory, multipotent cells, distinguishing them from adjacent stationary neural progenitors in the neurepithelium. Histone methylation spatiotemporally regulates neural crest gene expression; however, the protein methyltransferases active in neural crest precursors are unknown. Moreover, the regulation of methylation during the dynamic process of neural crest migration is unclear. Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells. NSD3 expression commences before up-regulation of neural crest genes, and NSD3 is necessary for expression of the neural plate border gene Msx1, as well as the key neural crest transcription factors Sox10, Snail2, Sox9, and FoxD3, but not gene expression generally. Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation. In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration. These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration. PMID:25318671

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

PubMed

Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

2016-08-01

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20

PubMed Central

Yao, Li; Yu, Lin-Lu; Zhang, Jun-Jie; Xie, Xiang-Ting; Tao, Qing; Yan, Xin; Hong, Qing; Qiu, Ji-Guo

2016-01-01

ABSTRACT Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt. A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. IMPORTANCE Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba

MoSET1 (Histone H3K4 Methyltransferase in Magnaporthe oryzae) Regulates Global Gene Expression during Infection-Related Morphogenesis

PubMed Central

Pham, Kieu Thi Minh; Inoue, Yoshihiro; Vu, Ba Van; Nguyen, Hanh Hieu; Nakayashiki, Toru; Ikeda, Ken-ichi; Nakayashiki, Hitoshi

2015-01-01

Here we report the genetic analyses of histone lysine methyltransferase (KMT) genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1) impaired in histone H3 lysine 4 methylation (H3K4me) showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3) and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1) Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2) In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3) Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes), and during infection-related morphogenesis (1,385 genes), indicating that MoSET1 has a role in gene repression either directly or more

Jasmonic acid carboxyl methyltransferase regulates development and herbivory-induced defense response in rice.

PubMed

Qi, Jinfeng; Li, Jiancai; Han, Xiu; Li, Ran; Wu, Jianqiang; Yu, Haixin; Hu, Lingfei; Xiao, Yutao; Lu, Jing; Lou, Yonggen

2016-06-01

Jasmonic acid (JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase (JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate (MeJA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene, OsJMT1, whose encoding protein was localized in the cytosol, we found that the recombinant OsJMT1 protein catalyzed JA to MeJA. OsJMT1 is up-regulated in response to infestation with the brown planthopper (BPH; Nilaparvata lugens). Plants in which OsJMT1 had been overexpressed (oe-JMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased MeJA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine (JA-Ile). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs, probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H2 O2 and MeJA in oe-JMT plants. These results indicate that OsJMT1, by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice. © 2015 Institute of Botany, Chinese Academy of Sciences.

Association of codon 108/158 catechol-O-methyltransferase gene polymorphism with the psychiatric manifestations of velo-cardio-facial syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lachman, H.M.; Papolos, D.F.; Veit, S.

Velo-cardio-facial-syndrome (VCFS) is a common congenital disorder associated with typical facial appearance, cleft palate, cardiac defects, and learning disabilities. The majority of patients have an interstitial deletion on chromosome 22q11. In addition to physical abnormalities, a variety of psychiatric illnesses have been reported in patients with VCFS, including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. The psychiatric manifestations of VCFS could be due to haploinsufficiency of a gene(s) within 22q11. One candidate that has been mapped to this region is catechol-O-methyltransferase (COMT). We recently identified a polymorphism in the COMT gene that leads to a valine{r_arrow}methionine substitution at aminomore » acid 158 of the membrane-bound form of the enzyme. Homozygosity for COMT158{sup met} leads to a 3- to 4-fold reduction in enzymatic activity, compared with homozygotes for COMT158{sup met}. We now report that in a population of patients with VCFS, there is an apparent association between the low-activity allele, COMT158{sup met}, and the development of bipolar spectrum disorder, and in particular, a rapid-cycling form. 33 refs., 3 tabs.« less

Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability

PubMed Central

Bernkopf, Marie; Webersinke, Gerald; Tongsook, Chanakan; Koyani, Chintan N.; Rafiq, Muhammad A.; Ayaz, Muhammad; Müller, Doris; Enzinger, Christian; Aslam, Muhammad; Naeem, Farooq; Schmidt, Kurt; Gruber, Karl; Speicher, Michael R.; Malle, Ernst; Macheroux, Peter; Ayub, Muhammad; Vincent, John B.; Windpassinger, Christian; Duba, Hans-Christoph

2014-01-01

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development. PMID:24626631

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

PubMed

Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

PubMed

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Chemical Probes of Histone Lysine Methyltransferases

PubMed Central

2015-01-01

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

PubMed Central

2012-01-01

Background Silene latifolia and its pollinator, the noctuid moth Hadena bicruris, represent an open nursery pollination system wherein floral volatiles, especially veratrole (1, 2-dimethoxybenzene), lilac aldehydes, and phenylacetaldehyde are of key importance for floral signaling. Despite the important role of floral scent in ensuring reproductive success in S. latifolia, the molecular basis of scent biosynthesis in this species has not yet been investigated. Results We isolated two full-length cDNAs from S. latifolia that show similarity to rose orcinol O-methyltransferase. Biochemical analysis showed that both S. latifolia guaiacol O-methyltransferase1 (SlGOMT1) &S. latifolia guaiacol O-methyltransferase2 (SlGOMT2) encode proteins that catalyze the methylation of guaiacol to form veratrole. A large Km value difference between SlGOMT1 (~10 μM) and SlGOMT2 (~501 μM) resulted that SlGOMT1 is 31-fold more catalytically efficient than SlGOMT2. qRT-PCR expression analysis showed that the SlGOMT genes are specifically expressed in flowers and male S. latifolia flowers had 3- to 4-folds higher level of GOMT gene transcripts than female flower tissues. Two related cDNAs, S. dioica O-methyltransferase1 (SdOMT1) and S. dioica O-methyltransferase2 (SdOMT2), were also obtained from the sister species Silene dioica, but the proteins they encode did not methylate guaiacol, consistent with the lack of veratrole emission in the flowers of this species. Our evolutionary analysis uncovered that SlGOMT1 and SlGOMT2 genes evolved under positive selection, whereas SdOMT1 and SdOMT2 genes show no evidence for selection. Conclusions Altogether, we report the identification and functional characterization of the gene, SlGOMT1 that efficiently catalyzes veratrole formation, whereas another copy of this gene with only one amino acid difference, SlGOMT2 was found to be less efficient for veratrole synthesis in S. latifolia. PMID:22937972

Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms

PubMed Central

Jiang, Weili; Shang, Siyuan; Su, Yanjie

2015-01-01

People may experience an “aha” moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving. PMID:26528222

Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms.

PubMed

Jiang, Weili; Shang, Siyuan; Su, Yanjie

2015-01-01

People may experience an "aha" moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving.

Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability.

PubMed

Bernkopf, Marie; Webersinke, Gerald; Tongsook, Chanakan; Koyani, Chintan N; Rafiq, Muhammad A; Ayaz, Muhammad; Müller, Doris; Enzinger, Christian; Aslam, Muhammad; Naeem, Farooq; Schmidt, Kurt; Gruber, Karl; Speicher, Michael R; Malle, Ernst; Macheroux, Peter; Ayub, Muhammad; Vincent, John B; Windpassinger, Christian; Duba, Hans-Christoph

2014-08-01

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development. © The Author 2014. Published by Oxford University Press.

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

PubMed Central

Nield, Blair S.; Willows, Robert D.; Torda, Andrew E.; Gillings, Michael R.; Holmes, Andrew J.; Nevalainen, K.M. Helena; Stokes, H.W.; Mabbutt, Bridget C.

2004-01-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%–28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins. PMID:15152095

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase.

PubMed

Nield, Blair S; Willows, Robert D; Torda, Andrew E; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Mabbutt, Bridget C

2004-06-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

PubMed

Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

2017-01-01

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea*S⃞

PubMed Central

Ferguson, Tsuneo; Soares, Jitesh A.; Lienard, Tanja; Gottschalk, Gerhard; Krzycki, Joseph A.

2009-01-01

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function. PMID:19043046

Characterization of two in vivo-expressed methyltransferases of the Mycobacterium tuberculosis complex: antigenicity and genetic regulation

PubMed Central

Golby, Paul; Nunez, Javier; Cockle, Paul J.; Ewer, Katie; Logan, Karen; Hogarth, Philip; Vordermeier, H. Martin; Hinds, Jason; Hewinson, R. Glyn; Gordon, Stephen V.

2011-01-01

Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli. PMID:18375799

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon

PubMed Central

2013-01-01

Background Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. Results We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. Conclusion These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency. PMID:23902793

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

PubMed

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

PubMed Central

Motorin, Y; Grosjean, H

1999-01-01

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w. PMID:10445884

Nomenclature for alleles of the thiopurine methyltransferase gene

PubMed Central

Appell, Malin L.; Berg, Jonathan; Duley, John; Evans, William E.; Kennedy, Martin A.; Lennard, Lynne; Marinaki, Tony; McLeod, Howard L.; Relling, Mary V.; Schaeffeler, Elke; Schwab, Matthias; Weinshilboum, Richard; Yeoh, Allen E.J.; McDonagh, Ellen M.; Hebert, Joan M.; Klein, Teri E.; Coulthard, Sally A.

2013-01-01

The drug-metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine, and azathioprine, drugs that are widely used for treatment of acute leukemias, inflammatory bowel diseases, and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants that cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pretreatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however, this has caused some problems as exemplified by two instances in which authors’ articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define the nomenclature and numbering of novel variants for the TPMT gene. A website (http://www.imh.liu.se/tpmtalleles) serves as a platform for this work. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (G > A) from TPMT*24 to TPMT*30 and position 611 (T > C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged. PMID:23407052

Molecular cloning and characterization of tetrahydroprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis in opium poppy.

PubMed

Liscombe, David K; Facchini, Peter J

2007-05-18

S-Adenosyl-l-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K(m) values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 microm, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

Rice putative methyltransferase gene OsTSD2 is required for root development involving pectin modification

PubMed Central

Qu, Lianghuan; Wu, Chunyan; Zhang, Fei; Wu, Yangyang; Fang, Chuanying; Jin, Cheng; Liu, Xianqing; Luo, Jie

2016-01-01

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Here, we report the functional characterization of the OsTSD2 gene, which encodes a putative methyltransferase in rice. All three independent T-DNA insertion lines of OsTSD2 displayed dwarf phenotypes and serial alterations in different zones of the root. These alterations included abnormal cellular adhesion and schizogenous aerenchyma formation in the meristematic zone, inhibited root elongation in the elongation zone, and higher lateral root density in the mature zone. Immunofluorescence (with LM19) and Ruthenium Red staining of the roots showed that unesterified homogalacturonan (HG) was increased in Ostsd2 mutants. Biochemical analysis of cell wall pectin polysaccharides revealed that both the monosaccharide composition and the uronic acid content were decreased in Ostsd2 mutants. Increased endogenous ABA content and opposite roles performed by ABA and IAA in regulating cellular adhesion in the Ostsd2 mutants suggested that OsTSD2 is required for root development in rice through a pathway involving pectin synthesis/modification. A hypothesis to explain the relationship among OsTSD2, pectin methylesterification, and root development is proposed, based on pectin’s function in regional cell extension/division in a zone-dependent manner. PMID:27497286

Synthesis of Lysine Methyltransferase Inhibitors

NASA Astrophysics Data System (ADS)

Ye, Tao; Hui, Chunngai

2015-07-01

Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

PubMed Central

Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

2010-01-01

N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

PubMed

Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

2007-02-23

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

Comparative effects of kainic, quisqualic, and ibotenic acids on phenylethanolamine-N-methyltransferase-containing cells of rat retina.

PubMed

Cohen, J

1989-02-01

Phenylethanolamine-N-methyltransferase (PNMT) activity is located in a subpopulation of amacrine cells in the inner nuclear layer of the rat retina. Kainic, quisqualic, and ibotenic acids, all of which are analogues of glutamic acid, were injected intravitreally to the right and saline to the contralateral left eyes of adult male rats in order to determine the effect of these agents upon retinal PNMT activity. Animals were sacrificed 1 week later for tissue removal. The effect of these agents was measured by radiometric assay for PNMT. The fall in PNMT activity was used to measure the sensitivity of the PNMT-containing cells to these agents. Kainic acid was the most potent, producing the greatest reduction in PNMT activity in the smallest doses. Quisqualic acid was intermediate in potency to that of kainic and ibotenic acids. Ibotenic acid reduced PNMT activity only in extremely high doses. The PNMT-containing cells are sensitive to the toxic actions of kainic and quisqualic acids, but relatively insensitive to the actions of ibotenic acid.

Structural characterization of the mitomycin 7-O-methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Shanteri; Chang, Aram; Goff, Randal D.

2014-10-02

Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylationmore » to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.« less

The Role of the Catechol-o-methyltransferase (COMT) Gene Val158Met in Aggressive Behavior, A Review of Genetic Studies

PubMed Central

Qayyum, Arqam; Zai, Clement C.; Hirata, Yuko; Tiwari, Arun K.; Cheema, Sheraz; Nowrouzi, Behdin; Beitchman, Joseph H.; Kennedy, James L.

2015-01-01

Aggressive behaviors have become a major public health problem, and early-onset aggression can lead to outcomes such as substance abuse, antisocial personality disorder among other issues. In recent years, there has been an increase in research in the molecular and genetic underpinnings of aggressive behavior, and one of the candidate genes codes for the catechol-O-methyltransferase (COMT). COMT is involved in catabolizing catecholamines such as dopamine. These neurotransmitters appear to be involved in regulating mood which can contribute to aggression. The most common gene variant studied in the COMT gene is the Valine (Val) to Methionine (Met) substitution at codon 158. We will be reviewing the current literature on this gene variant in aggressive behavior. PMID:26630958

Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordii.

PubMed

Guan, Hongyu; Zhao, Yujun; Su, Ping; Tong, Yuru; Liu, Yujia; Hu, Tianyuan; Zhang, Yifeng; Zhang, Xianan; Li, Jia; Wu, Xiaoyi; Huang, Luqi; Gao, Wei

2017-09-01

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii ( TwSMT1 ). TwSMT1 (GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli ( E. coli ) and showed SMT activity. The expression of TwSMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803*S⃞

PubMed Central

Kopycki, Jakub Grzegorz; Stubbs, Milton T.; Brandt, Wolfgang; Hagemann, Martin; Porzel, Andrea; Schmidt, Jürgen; Schliemann, Willibald; Zenk, Meinhart H.; Vogt, Thomas

2008-01-01

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed. PMID:18502765

Increased phosphatidylethanolamine N-methyltransferase gene expression in non-small-cell lung cancer tissue predicts shorter patient survival

PubMed Central

ZINRAJH, DAVID; HÖRL, GERD; JÜRGENS, GÜNTHER; MARC, JANJA; SOK, MIHA; CERNE, DARKO

2014-01-01

Lipid mobilization is of great importance for tumor growth and studies have suggested that cancer cells exhibit abnormal choline phospholipid metabolism. In the present study, we hypothesized that phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is increased in non-small-cell lung cancer (NSCLC) tissues and that increased gene expression acts as a predictor of shorter patient survival. Forty-two consecutive patients with resected NSCLC were enrolled in this study. Paired samples of lung cancer tissues and adjacent non-cancer lung tissues were collected from resected specimens for the estimation of PEMT expression. SYBR Green-based real-time polymerase chain reaction was used for quantification of PEMT mRNA in lung cancer tissues. Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activities had already been measured in the same tissues. During a four-year follow-up, 21 patients succumbed to tumor progression. One patient did not survive due to non-cancer reasons and was not included in the analysis. Cox regression analysis was used to assess the prognostic value of PEMT expression. Our findings show that elevated PEMT expression in the cancer tissue, relative to that in the adjacent non-cancer lung tissue, predicts shorter patient survival independently of standard prognostic factors and also independently of increased LPL or FASN activity, the two other lipid-related predictors of shorter patient survival. These findings suggest that active phosphatidylcholine and/or choline metabolism are essential for tumor growth and progression. PMID:24932311

Increased phosphatidylethanolamine N-methyltransferase gene expression in non-small-cell lung cancer tissue predicts shorter patient survival.

PubMed

Zinrajh, David; Hörl, Gerd; Jürgens, Günther; Marc, Janja; Sok, Miha; Cerne, Darko

2014-06-01

Lipid mobilization is of great importance for tumor growth and studies have suggested that cancer cells exhibit abnormal choline phospholipid metabolism. In the present study, we hypothesized that phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is increased in non-small-cell lung cancer (NSCLC) tissues and that increased gene expression acts as a predictor of shorter patient survival. Forty-two consecutive patients with resected NSCLC were enrolled in this study. Paired samples of lung cancer tissues and adjacent non-cancer lung tissues were collected from resected specimens for the estimation of PEMT expression. SYBR Green-based real-time polymerase chain reaction was used for quantification of PEMT mRNA in lung cancer tissues. Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activities had already been measured in the same tissues. During a four-year follow-up, 21 patients succumbed to tumor progression. One patient did not survive due to non-cancer reasons and was not included in the analysis. Cox regression analysis was used to assess the prognostic value of PEMT expression. Our findings show that elevated PEMT expression in the cancer tissue, relative to that in the adjacent non-cancer lung tissue, predicts shorter patient survival independently of standard prognostic factors and also independently of increased LPL or FASN activity, the two other lipid-related predictors of shorter patient survival. These findings suggest that active phosphatidylcholine and/or choline metabolism are essential for tumor growth and progression.

A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Nan; Ferrer, Jean-Luc; Moon, Hong S

2012-01-01

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested formore » methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.« less

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

DOE PAGES

Ticak, Tomislav; Kountz, D. J.; Girosky, K. E.; ...

2014-10-13

COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptorsmore » such as nitrate or fumarate, producing dimethylglycine and CO 2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. Additionally, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. Lastly, they are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.« less

Rice putative methyltransferase gene OsTSD2 is required for root development involving pectin modification.

PubMed

Qu, Lianghuan; Wu, Chunyan; Zhang, Fei; Wu, Yangyang; Fang, Chuanying; Jin, Cheng; Liu, Xianqing; Luo, Jie

2016-10-01

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Here, we report the functional characterization of the OsTSD2 gene, which encodes a putative methyltransferase in rice. All three independent T-DNA insertion lines of OsTSD2 displayed dwarf phenotypes and serial alterations in different zones of the root. These alterations included abnormal cellular adhesion and schizogenous aerenchyma formation in the meristematic zone, inhibited root elongation in the elongation zone, and higher lateral root density in the mature zone. Immunofluorescence (with LM19) and Ruthenium Red staining of the roots showed that unesterified homogalacturonan (HG) was increased in Ostsd2 mutants. Biochemical analysis of cell wall pectin polysaccharides revealed that both the monosaccharide composition and the uronic acid content were decreased in Ostsd2 mutants. Increased endogenous ABA content and opposite roles performed by ABA and IAA in regulating cellular adhesion in the Ostsd2 mutants suggested that OsTSD2 is required for root development in rice through a pathway involving pectin synthesis/modification. A hypothesis to explain the relationship among OsTSD2, pectin methylesterification, and root development is proposed, based on pectin's function in regional cell extension/division in a zone-dependent manner. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations

USDA-ARS?s Scientific Manuscript database

With S-adenosylmethionine (SAM) acting as the methyl donor, caffeic acid O-methyltransferase from Sorghum bicolor (SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde, to form sinapaldehyde. In order to determine the mechanism of SbCOMT and understand the red...

Polymorphisms of the thiopurine S-methyltransferase gene among the Libyan population

PubMed Central

Zeglam, Hamza Ben; Benhamer, Abdrazak; Aboud, Adel; Rtemi, Haitem; Mattardi, Meftah; Saleh, Saleh Suleiman; Bashein, Abdullah; Enattah, Nabil

2015-01-01

Background Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of 6-mercaptopurine and azathioprine. Low activity phenotypes are correlated with polymorphism in the TPMT gene. Patients with low or undetectable TMPT activity could develop severe myelosuppression when they are treated with standard doses of thiopurine drugs. Since ethnic differences in the TPMT gene polymorphism have been demonstrated worldwide, assessing it in the Libyan population is worthwhile. Methods We investigated TPMT gene polymorphism in a total of 246 Libyan healthy adult blood donors from three different Libyan regions (Tripoli, Yefren, and Tawargha) and 50 children with acute lymphoblastic leukaemia (ALL). We used polymerase chain reaction restriction length polymorphism (PCR-RFLP) and allele-specific PCR-based assays to analyse the TPMT gene for the variants *2 c.238 G>C, *3A (c.460 G>A and c.719 A>G), *3B (c.460 G>A), and *3C (c.719 A>G). Results Our results show that the TPMT variants associated with low enzymatic activity were detected in 3.25% (8 in 246) of adult Libyan individuals and the frequency of total mutant alleles was 1.63%. Heterozygous genotypes were TPMT*3A in three subjects (0.61%) and TPMT*3C in five subjects (1.02%). No TPMT*2 and TPMT*3B allelic variants and no homozygous or compound heterozygous mutant alleles were detected. The normal allele (wild-type) was found in 98.4% of the adult individuals studied. No mutant alleles were detected among the 50 children who had ALL. Conclusions We report on the presence of the TPMT*3C and *3A mutant alleles in the Libyan population. Therefore, monitoring the patients to be treated with doses of thiopurine drugs for TPMT variants is worthwhile to avoid the development of severe myelosuppression. PMID:25819542

Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimura, Shuhei; Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574; Sawatsubashi, Shun

2008-07-11

Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressedmore » EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.« less

Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1*

PubMed Central

Lee, Byron H.; Yegnasubramanian, Srinivasan; Lin, Xiaohui; Nelson, William G.

2007-01-01

CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycyti-dine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer. PMID:16230360

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

PubMed

Ortega-Molina, Ana; Boss, Isaac W; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A; Gascoyne, Randy D; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M; Wendel, Hans-Guido

2015-10-01

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids.

PubMed

Mahesh, Venkataramaiah; Rakotomalala, Jean Jacques; Le Gal, Lénaïg; Vigne, Hélène; de Kochko, Alexandre; Hamon, Serge; Noirot, Michel; Campa, Claudine

2006-09-01

Biosynthesis of caffeoylquinic acids occurs via the phenylpropanoid pathway in which the phenylalanine ammonia-lyase (PAL) acts as a key-control enzyme. A full-length cDNA (pF6), corresponding to a PAL gene (CcPAL1), was isolated by screening a Coffea canephora fruit cDNA library and its corresponding genomic sequence was characterized. Amplification of total DNA from seven Coffea species revealed differences in intronic length. This interspecific polymorphism was used to locate the gene on a genetic map established for a backcross progeny between Coffea pseudozanguebariae and C. dewevrei. The CcPAL1 gene was found on the same linkage group, but genetically independent, as a caffeoyl-coenzyme A-O-methyltransferase gene, another gene intervening in the phenylpropanoid pathway. In the same backcross, a lower caffeoylquinic acid content was observed in seeds harvested from plants harbouring the C. pseudozanguebariae CcPAL1 allele. Involvement of the CcPAL1 allelic form in the differential accumulation of caffeoylquinic acids in coffee green beans is then discussed.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

PubMed

Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

2018-01-01

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones.

PubMed

Chaiprasongsuk, Minta; Zhang, Chi; Qian, Ping; Chen, Xinlu; Li, Guanglin; Trigiano, Robert N; Guo, Hong; Chen, Feng

2018-05-01

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms. Copyright © 2018 Elsevier Ltd. All rights reserved.

Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii.

PubMed

Pampa, K J; Madan Kumar, S; Hema, M K; Kumara, Karthik; Naveen, S; Kunishima, Naoki; Lokanath, N K

2017-12-01

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

Exon resequencing of H3K9 methyltransferase complex genes, EHMT1, EHTM2 and WIZ, in Japanese autism subjects.

PubMed

Balan, Shabeesh; Iwayama, Yoshimi; Maekawa, Motoko; Toyota, Tomoko; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Esaki, Kayoko; Yamada, Kazuo; Iwata, Yasuhide; Suzuki, Katsuaki; Ide, Masayuki; Ota, Motonori; Fukuchi, Satoshi; Tsujii, Masatsugu; Mori, Norio; Shinkai, Yoichi; Yoshikawa, Takeo

2014-01-01

Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD. Since G9a-GLP-Wiz forms a heteromeric methyltransferase complex, all the protein-coding regions and exon/intron boundaries of EHMT1, EHMT2 and WIZ were sequenced in Japanese ASD subjects. The detected variants were prioritized based on novelty and functionality. The expression levels of these genes were tested in blood cells and postmortem brain samples from ASD and control subjects. Expression of EHMT1 and EHMT2 isoforms were determined by digital PCR. We identified six nonsynonymous variants: three in EHMT1, two in EHMT2 and one in WIZ. Two variants, the EHMT1 ankyrin repeat domain (Lys968Arg) and EHMT2 SET domain (Thr961Ile) variants were present exclusively in cases, but showed no statistically significant association with ASD. The EHMT2 transcript expression was significantly elevated in the peripheral blood cells of ASD when compared with control samples; but not for EHMT1 and WIZ. Gene expression levels of EHMT1, EHMT2 and WIZ in Brodmann area (BA) 9, BA21, BA40 and the dorsal raphe nucleus (DoRN) regions from postmortem brain samples showed no significant changes between ASD and control subjects. Nor did expression levels of EHMT1 and EHMT2 isoforms in the prefrontal cortex differ significantly between ASD and

IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

EPA Science Inventory

Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

Molecular variation and horizontal gene transfer of the homocysteine methyltransferase gene mmuM and its distribution in clinical pathogens.

PubMed

Ying, Jianchao; Wang, Huifeng; Bao, Bokan; Zhang, Ying; Zhang, Jinfang; Zhang, Cheng; Li, Aifang; Lu, Junwan; Li, Peizhen; Ying, Jun; Liu, Qi; Xu, Teng; Yi, Huiguang; Li, Jinsong; Zhou, Li; Zhou, Tieli; Xu, Zuyuan; Ni, Liyan; Bao, Qiyu

2015-01-01

The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.

Preliminary individualized chemotherapy for malignant astrocytomas based on O6-methylguanine-deoxyribonucleic acid methyltransferase methylation analysis.

PubMed

Watanabe, Takao; Katayama, Yoichi; Ogino, Akiyoshi; Ohta, Takashi; Yoshino, Atsuo; Fukushima, Takao

2006-08-01

O(6)-methylguanine-deoxyribonucleic acid methyltransferase gene (MGMT) methylation is apparently correlated with responsiveness to nitrosourea chemotherapy, suggesting this alkylating agent should be effective against MGMT-methylated tumors. MGMT appears not to be linked to platinum resistance, so platinum chemotherapy should be used for MGMT-unmethylated tumors. This study was a preliminary trial of individualized chemotherapy based on MGMT methylation status in a total of 20 patients with newly diagnosed malignant astrocytomas (9 anaplastic astrocytomas and 11 glioblastomas multiforme). The procarbazine, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea, and vincristine (PAV) regimen was administered to seven patients with MGMT-methylated tumors, and the carboplatin and etoposide (CE) regimen was administered to 13 patients with MGMT-unmethylated tumors. Objective response to the PAV therapy was noted in all three patients with measurable residual tumor (2 complete responses and 1 partial response). Five of the seven patients continued to be disease-free after initiation of the PAV therapy. Objective response to the CE therapy was seen in only one of seven patients with measurable residual tumor (1 partial response). Three of the 13 patients were free from progression, whereas the remaining 10 patients showed early progression. The PAV regimen is effective against MGMT-methylated malignant astrocytomas, but the CE regimen is not useful at the given dose and schedule in MGMT-unmethylated tumors.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development

PubMed Central

Ortega-Molina, Ana; Boss, Isaac W.; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W.; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A.; Gascoyne, Randy D.; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M.; Wendel, Hans-Guido

2015-01-01

The lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center (GC) involution, impedes B cell differentiation and class switch recombination (CSR). Integrative genomic analyses indicate that KMT2D affects H3K4 methylation and expression of a specific set of genes including those in the CD40, JAK-STAT, Toll-like receptor, and B cell receptor pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3, and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell activating pathways. PMID:26366710

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yuan; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3; Wu, Keqiang

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants.more » We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.« less

PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity.

PubMed

Miranda, Tina Branscombe; Miranda, Mark; Frankel, Adam; Clarke, Steven

2004-05-28

We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.

Arabidopsis histone methyltransferase SET DOMAIN GROUP8 mediates induction of the jasmonate/ethylene pathway genes in plant defense response to necrotrophic fungi.

PubMed

Berr, Alexandre; McCallum, Emily J; Alioua, Abdelmalek; Heintz, Dimitri; Heitz, Thierry; Shen, Wen-Hui

2010-11-01

As sessile organisms, plants have to endure a wide variety of biotic and abiotic stresses, and accordingly they have evolved intricate and rapidly inducible defense strategies associated with the activation of a battery of genes. Among other mechanisms, changes in chromatin structure are thought to provide a flexible, global, and stable means for the regulation of gene transcription. In support of this idea, we demonstrate here that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) plays a crucial role in plant defense against fungal pathogens by regulating a subset of genes within the jasmonic acid (JA) and/or ethylene signaling pathway. We show that the loss-of-function mutant sdg8-1 displays reduced resistance to the necrotrophic fungal pathogens Alternaria brassicicola and Botrytis cinerea. While levels of JA, a primary phytohormone involved in plant defense, and camalexin, a major phytoalexin against fungal pathogens, remain unchanged or even above normal in sdg8-1, induction of several defense genes within the JA/ethylene signaling pathway is severely compromised in response to fungal infection or JA treatment in mutant plants. Both downstream genes and, remarkably, also upstream mitogen-activated protein kinase kinase genes MKK3 and MKK5 are misregulated in sdg8-1. Accordingly, chromatin immunoprecipitation analysis shows that sdg8-1 impairs dynamic changes of histone H3 lysine 36 methylation at defense marker genes as well as at MKK3 and MKK5, which normally occurs upon infection with fungal pathogens or methyl JA treatment in wild-type plants. Our data indicate that SDG8-mediated histone H3 lysine 36 methylation may serve as a memory of permissive transcription for a subset of defense genes, allowing rapid establishment of transcriptional induction.

Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

PubMed

Kawamura, Takuya; Anraku, Ryou; Hasegawa, Takahiro; Tomikawa, Chie; Hori, Hiroyuki

2014-12-23

We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama).

PubMed

Van Ekert, Evelien; Shatters, Robert G; Rougé, Pierre; Powell, Charles A; Smagghe, Guy; Borovsky, Dov

2015-01-01

The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus 'Liberibacter' asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10(-3) and 0.217 × 10(-3) s(-1), respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10(-3), 0.013 × 10(-3), and 0.003 × 10(-3) s(-1), respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca(2+), Mg(2+) or Zn(2+), however, Zn(2+) (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.).

PubMed

Brenner, Everton A; Zein, Imad; Chen, Yongsheng; Andersen, Jeppe R; Wenzel, Gerhard; Ouzunova, Milena; Eder, Joachim; Darnhofer, Birte; Frei, Uschi; Barrière, Yves; Lübberstedt, Thomas

2010-02-12

OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

PubMed Central

2010-01-01

Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Conclusions Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies. PMID:20152036

Identification of phosphomethylethanolamine N-methyltransferase from Arabidopsis and its role in choline and phospholipid metabolism.

PubMed

BeGora, Michael D; Macleod, Mitchell J R; McCarry, Brian E; Summers, Peter S; Weretilnyk, Elizabeth A

2010-09-17

Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-l-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.

Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian P

2015-01-02

Ergothioneine is an N-α-trimethyl-2-thiohistidine derivative that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox-active betaine starts with trimethylation of the α-amino group of histidine. The three consecutive methyl transfers are catalyzed by the S-adenosylmethionine-dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N-α-dimethylhistidine and S-adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 10(7)-fold and transform the histidine-methyltransferase into a proficient tryptophan-methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Folic Acid Supplementation Delays Atherosclerotic Lesion Development by Modulating MCP1 and VEGF DNA Methylation Levels In Vivo and In Vitro

PubMed Central

Cui, Shanshan; Li, Wen; Lv, Xin; Wang, Pengyan; Gao, Yuxia; Huang, Guowei

2017-01-01

The pathogenesis of atherosclerosis has been partly acknowledged to result from aberrant epigenetic mechanisms. Accordingly, low folate levels are considered to be a contributing factor to promoting vascular disease because of deregulation of DNA methylation. We hypothesized that increasing the levels of folic acid may act via an epigenetic gene silencing mechanism to ameliorate atherosclerosis. Here, we investigated the atheroprotective effects of folic acid and the resultant methylation status in high-fat diet-fed ApoE knockout mice and in oxidized low-density lipoprotein-treated human umbilical vein endothelial cells. We analyzed atherosclerotic lesion histology, folate concentration, homocysteine concentration, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and DNA methyltransferase activity, as well as monocyte chemotactic protein-1 (MCP1) and vascular endothelial growth factor (VEGF) expression and promoter methylation. Folic acid reduced atherosclerotic lesion size in ApoE knockout mice. The underlying folic acid protective mechanism appears to operate through regulating the normal homocysteine state, upregulating the SAM: SAH ratio, elevating DNA methyltransferase activity and expression, altering MCP1 and VEGF promoter methylation, and inhibiting MCP1 and VEGF expression. We conclude that folic acid supplementation effectively prevented atherosclerosis by modifying DNA methylation through the methionine cycle, improving DNA methyltransferase activity and expression, and thus changing the expression of atherosclerosis-related genes. PMID:28475147

The catechol-O-methyltransferase gene (COMT) and cognitive function from childhood through adolescence

PubMed Central

Gaysina, Darya; Xu, Man K.; Barnett, Jennifer H.; Croudace, Tim J.; Wong, Andrew; Richards, Marcus; Jones, Peter B.

2013-01-01

Genetic variation in the catechol-O-methyltransferase gene (COMT) can influence cognitive function, and this effect may depend on developmental stage. Using a large representative British birth cohort, we investigated the effect of COMT on cognitive function (verbal and non-verbal) at ages 8 and 15 years taking into account the possible modifying effect of pubertal stage. Five functional COMT polymorphisms, rs6269, rs4818, rs4680, rs737865 and rs165599 were analysed. Associations between COMT polymorphisms and cognition were tested using regression and latent variable structural equation modelling (SEM). Before correction for multiple testing, COMT rs737865 showed association with reading comprehension, verbal ability and global cognition at age 15 years in pubescent boys only. Although there was some evidence for age- and sex-specific effects of the COMT rs737865 none remained significant after correction for multiple testing. Further studies are necessary in order to make firmer conclusions. PMID:23178897

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

PubMed

Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

2015-10-01

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto�

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

PubMed Central

Levac, Dylan; Cázares, Paulo; Yu, Fang

2016-01-01

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene.

PubMed

Matsukuma, S; Nakatsuru, Y; Nakagawa, K; Utakoji, T; Sugano, H; Kataoka, H; Sekiguchi, M; Ishikawa, T

1989-11-01

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes. Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.

PubMed

Tasseva, Guergana; van der Veen, Jelske N; Lingrell, Susanne; Jacobs, René L; Vance, Dennis E; Vance, Jean E

2016-02-01

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

PubMed

Al-Hadid, Qais; White, Jonelle; Clarke, Steven

2016-02-12

A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel arsenic methyltransferase gene of Westerdykella aurantiaca isolated from arsenic contaminated soil: phylogenetic, physiological, and biochemical studies and its role in arsenic bioremediation.

PubMed

Verma, Shikha; Verma, Pankaj Kumar; Meher, Alok Kumar; Dwivedi, Sanjay; Bansiwal, Amit Kumar; Pande, Veena; Srivastava, Pankaj Kumar; Verma, Praveen Chandra; Tripathi, Rudra Deo; Chakrabarty, Debasis

2016-03-01

Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks.

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia

PubMed Central

Du, Hui; Wu, Jie; Ji, Kui-Xian; Zeng, Qing-Yin; Bhuiya, Mohammad-Wadud; Su, Shang; Shu, Qing-Yan; Ren, Hong-Xu; Liu, Zheng-An; Wang, Liang-Sheng

2015-01-01

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp. PMID:26208646

Catechol-O-methyltransferase gene haplotypes in Mexican and Spanish patients with fibromyalgia

PubMed Central

Vargas-Alarcón, Gilberto; Fragoso, José-Manuel; Cruz-Robles, David; Vargas, Angélica; Vargas, Alfonso; Lao-Villadóniga, José-Ignacio; García-Fructuoso, Ferrán; Ramos-Kuri, Manuel; Hernández, Fernando; Springall, Rashidi; Bojalil, Rafael; Vallejo, Maite; Martínez-Lavín, Manuel

2007-01-01

Autonomic dysfunction is frequent in patients with fibromyalgia (FM). Heart rate variability analyses have demonstrated signs of ongoing sympathetic hyperactivity. Catecholamines are sympathetic neurotransmitters. Catechol-O-methyltransferase (COMT), an enzyme, is the major catecholamine-clearing pathway. There are several single-nucleotide polymorphisms (SNPs) in the COMT gene associated with the different catecholamine-clearing abilities of the COMT enzyme. These SNPs are in linkage disequilibrium and segregate as 'haplotypes'. Healthy females with a particular COMT gene haplotype (ACCG) producing a defective enzyme are more sensitive to painful stimuli. The objective of our study was to define whether women with FM, from two different countries (Mexico and Spain), have the COMT gene haplotypes that have been previously associated with greater sensitivity to pain. All the individuals in the study were female. Fifty-seven Mexican patients and 78 Spanish patients were compared with their respective healthy control groups. All participants filled out the Fibromyalgia Impact Questionnaire (FIQ). Six COMT SNPs (rs2097903, rs6269, rs4633, rs4818, rs4680, and rs165599) were genotyped from peripheral blood DNA. In Spanish patients, there was a significant association between three SNPs (rs6269, rs4818, and rs4680) and the presence of FM when compared with healthy controls. Moreover, in Spanish patients with the 'high pain sensitivity' haplotype (ACCG), the disease, as assessed by the FIQ, was more severe. By contrast, Mexican patients displayed only a weak association between rs6269 and rs165599, and some FIQ subscales. In our group of Spanish patients, there was an association between FM and the COMT haplotype previously associated with high pain sensitivity. This association was not observed in Mexican patients. Studies with a larger sample size are needed in order to verify or amend these preliminary results. PMID:17961261

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

PubMed

Akhberdi, Oren; Zhang, Qian; Wang, Haichuan; Li, Yingying; Chen, Longfei; Wang, Dan; Yu, Xi; Wei, Dongsheng; Zhu, Xudong

2018-05-01

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi. Copyright © 2018 Elsevier GmbH. All rights reserved.

Identification of DNA Methyltransferase Genes in Human Pathogenic Bacteria by Comparative Genomics.

PubMed

Brambila-Tapia, Aniel Jessica Leticia; Poot-Hernández, Augusto Cesar; Perez-Rueda, Ernesto; Rodríguez-Vázquez, Katya

2016-06-01

DNA methylation plays an important role in gene expression and virulence in some pathogenic bacteria. In this report, we describe DNA methyltransferases (MTases) present in human pathogenic bacteria and compared them with related species, which are not pathogenic or less pathogenic, based in comparative genomics. We performed a search in the KEGG database of the KEGG database orthology groups associated with adenine and cytosine DNA MTase activities (EC: 2.1.1.37, EC: 2.1.1.113 and EC: 2.1.1.72) in 37 human pathogenic species and 18 non/less pathogenic relatives and performed comparisons of the number of these MTases sequences according to their genome size, the DNA MTase type and with their non-less pathogenic relatives. We observed that Helicobacter pylori and Neisseria spp. presented the highest number of MTases while ten different species did not present a predicted DNA MTase. We also detected a significant increase of adenine MTases over cytosine MTases (2.19 vs. 1.06, respectively, p < 0.001). Adenine MTases were the only MTases associated with restriction modification systems and DNA MTases associated with type I restriction modification systems were more numerous than those associated with type III restriction modification systems (0.84 vs. 0.17, p < 0.001); additionally, there was no correlation with the genome size and the total number of DNA MTases, indicating that the number of DNA MTases is related to the particular evolution and lifestyle of specific species, regulating the expression of virulence genes in some pathogenic bacteria.

Associations between arsenic (+3 oxidation state) methyltransferase (AS3MT) and N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) polymorphisms, arsenic metabolism, and cancer risk in a chilean population.

PubMed

de la Rosa, Rosemarie; Steinmaus, Craig; Akers, Nicholas K; Conde, Lucia; Ferreccio, Catterina; Kalman, David; Zhang, Kevin R; Skibola, Christine F; Smith, Allan H; Zhang, Luoping; Smith, Martyn T

2017-07-01

Inter-individual differences in arsenic metabolism have been linked to arsenic-related disease risks. Arsenic (+3) methyltransferase (AS3MT) is the primary enzyme involved in arsenic metabolism, and we previously demonstrated in vitro that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) also methylates the toxic inorganic arsenic (iAs) metabolite, monomethylarsonous acid (MMA), to the less toxic dimethylarsonic acid (DMA). Here, we evaluated whether AS3MT and N6AMT1 gene polymorphisms alter arsenic methylation and impact iAs-related cancer risks. We assessed AS3MT and N6AMT1 polymorphisms and urinary arsenic metabolites (%iAs, %MMA, %DMA) in 722 subjects from an arsenic-cancer case-control study in a uniquely exposed area in northern Chile. Polymorphisms were genotyped using a custom designed multiplex, ligation-dependent probe amplification (MLPA) assay for 6 AS3MT SNPs and 14 tag SNPs in the N6AMT1 gene. We found several AS3MT polymorphisms associated with both urinary arsenic metabolite profiles and cancer risk. For example, compared to wildtypes, individuals carrying minor alleles in AS3MT rs3740393 had lower %MMA (mean difference = -1.9%, 95% CI: -3.3, -0.4), higher %DMA (mean difference = 4.0%, 95% CI: 1.5, 6.5), and lower odds ratios for bladder (OR = 0.3; 95% CI: 0.1-0.6) and lung cancer (OR = 0.6; 95% CI: 0.2-1.1). Evidence of interaction was also observed for both lung and bladder cancer between these polymorphisms and elevated historical arsenic exposures. Clear associations were not seen for N6AMT1. These results are the first to demonstrate a direct association between AS3MT polymorphisms and arsenic-related internal cancer risk. This research could help identify subpopulations that are particularly vulnerable to arsenic-related disease. Environ. Mol. Mutagen. 58:411-422, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

PubMed Central

Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

2013-01-01

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site.

PubMed

van Bemmel, Dana M; Brank, Adam S; Eritja, Ramon; Marquez, Victor E; Christman, Judith K

2009-09-15

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

PubMed Central

van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

2009-01-01

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes.

PubMed Central

Møller, Michael T N; Samari, Hamid R; Fengsrud, Monica; Strømhaug, Per E; øStvold, Anne C; Seglen, Per O

2003-01-01

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels

The Histone H3 Lysine 9 Methyltransferase DIM-5 Modifies Chromatin at frequency and Represses Light-Activated Gene Expression

PubMed Central

Ruesch, Catherine E.; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S.; Zaman, Riasat; Joska, Tammy M.; Belden, William J.

2014-01-01

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1bd. These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. PMID:25429045

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

PubMed

Ruesch, Catherine E; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S; Zaman, Riasat; Joska, Tammy M; Belden, William J

2014-11-25

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. Copyright © 2015 Ruesch et al.

Investigating the genetic basis of theory of mind (ToM): the role of catechol-O-methyltransferase (COMT) gene polymorphisms.

PubMed

Xia, Haiwei; Wu, Nan; Su, Yanjie

2012-01-01

The ability to deduce other persons' mental states and emotions which has been termed 'theory of mind (ToM)' is highly heritable. First molecular genetic studies focused on some dopamine-related genes, while the genetic basis underlying different components of ToM (affective ToM and cognitive ToM) remain unknown. The current study tested 7 candidate polymorphisms (rs4680, rs4633, rs2020917, rs2239393, rs737865, rs174699 and rs59938883) on the catechol-O-methyltransferase (COMT) gene. We investigated how these polymorphisms relate to different components of ToM. 101 adults participated in our study; all were genetically unrelated, non-clinical and healthy Chinese subjects. Different ToM tasks were applied to detect their theory of mind ability. The results showed that the COMT gene rs2020917 and rs737865 SNPs were associated with cognitive ToM performance, while the COMT gene rs5993883 SNP was related to affective ToM, in which a significant gender-genotype interaction was found (p = 0.039). Our results highlighted the contribution of DA-related COMT gene on ToM performance. Moreover, we found out that the different SNP at the same gene relates to the discriminative aspect of ToM. Our research provides some preliminary evidence to the genetic basis of theory of mind which still awaits further studies.

Mechanism of activation of methyltransferases involved in translation by the Trm112 'hub' protein.

PubMed

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-08-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

PubMed

Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

2017-04-01

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia.

PubMed

Bronzini, I; Aresu, L; Paganin, M; Marchioretto, L; Comazzi, S; Cian, F; Riondato, F; Marconato, L; Martini, V; Te Kronnie, G

2017-09-01

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs. © 2016 John Wiley & Sons Ltd.

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation.

PubMed

Jelinic, Petar; Stehle, Jean-Christophe; Shaw, Phillip

2006-10-01

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation-DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

PubMed Central

Jelinic, Petar; Stehle, Jean-Christophe; Shaw, Phillip

2006-01-01

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation. PMID:17048991

Chromosomal 16S Ribosomal RNA Methyltransferase RmtE1 in Escherichia coli Sequence Type 448

PubMed Central

Li, Bin; Pacey, Marissa P.

2017-01-01

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone. PMID:28418308

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

PubMed Central

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H.; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-01-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases. PMID:21478168

Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

PubMed

Rosic, Nedeljka N

2012-04-01

Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases.

PubMed

Widiez, Thomas; Hartman, Thomas G; Dudai, Nativ; Yan, Qing; Lawton, Michael; Havkin-Frenkel, Daphna; Belanger, Faith C

2011-08-01

Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.

[Glycine-N-methyltransferase and Malignant Diseases of the Prostate].

PubMed

Heger, Z; Eckschlager, T; Stiborová, M; Adam, V

Prostate cancer (PC) constitutes a heterogeneous group of diseases with high prevalence rates that are still increasing, particularly in western countries. Since 1980, prostate specific antigen (PSA) and other diagnostic approaches have been used for PC screening; however, some of these approaches are often deemed painful and cause invasive damage of tissue. Therefore, molecular approaches to PC diagnosis are attracting increasing attention, potentially providing patients with less stressful situations and providing better diagnoses and even prognostic information. Recent metabolomic and genomic studies have suggested that biomolecules can be used as diagnostic or prognostic markers or as targets for the development of novel therapeutic modalities. One of these molecules is glycine-N-methyltransferase (GNMT), an enzyme that plays a pivotal role in the biochemical conversion of glycine to sarcosine. The link between this molecule (encoded by homonymous gene - GNMT) and PC has been confirmed at several levels, and thus GNMT can be considered a promising target for the development of advanced diagnostic and/or prognostic approaches. The aim of this study was to analyse the physiological role of GNMT and to examine in greater detail its connection with PC at different levels, including gene structure, gene expression, and metabolism, in which GNMT plays an important role, not only in controlling the methylation status of cells, but also the metabolism of folic acid and methionine. Last but not least, we discuss the importance of cellular methylation processes and the link between their aberrations and PC development.Key words: glycine - folic acid - metabolism - methylation - sarcosineThis work was supported by GA CR 16-18917S, League against Cancer Prague (project 2022015) and Czech Ministry of Health - RVO, UH Motol 00064203.The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.The Editorial Board

An open reading frame in intron seven of the sea urchin DNA-methyltransferase gene codes for a functional AP1 endonuclease.

PubMed

Cioffi, Anna Valentina; Ferrara, Diana; Cubellis, Maria Vittoria; Aniello, Francesco; Corrado, Marcella; Liguori, Francesca; Amoroso, Alessandro; Fucci, Laura; Branno, Margherita

2002-08-01

Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene. METEX expression is developmentally regulated, showing no correlation with DNA MTase expression. In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development. Two METEX cDNA clones (Met1 and Met2) that are different in the 3' end have been isolated in a cDNA library screening. The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure. The protein, overexpressed in E. coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity. Therefore the open reading frame, present in intron 7 of the P. lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

No association between catechol-O-methyltransferase polymorphisms and neurotic disorders among mainland Chinese university students.

PubMed

Kou, Changgui; Meng, Xiangfei; Xie, Bing; Shi, Jieping; Yu, Qiong; Yu, Yaqin; D'Arcy, Carl

2012-07-30

This study investigates the genetic association between catechol-O-methyltransferase (COMT) gene polymorphisms and neurotic disorders. Data were derived from a case-control association study of 255 undergraduates affected by neurotic disorders and 269 matched healthy undergraduate controls. The polymorphisms of eight tag single nucleotide polymorphisms (SNPs) on the COMT gene were tested using polymerase chain reaction (PCR)-based Ligase Detection Reaction (PCR-LDR). The eight tag SNPs on the COMT gene assessed were not associated with neurotic disorders. Our finding suggests that the COMT gene may not be a susceptibility gene for neurotic disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation.

PubMed

Thanh Mai Pham, Le; Kim, Yong Hwan

2016-01-01

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5'-adenosyl)-L-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones. Copyright © 2015 Elsevier Inc. All rights reserved.

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

PubMed Central

Sayegh, Joyce; Clarke, Steven G.

2008-01-01

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase. PMID:18515076

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

PubMed Central

Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1997-01-01

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

PubMed

Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

1997-05-13

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

QM/MM Free Energy Simulations of Salicylic Acid Methyltransferase: Effects of Stabilization of TS-like Structures on Substrate Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jianzhuang; Xu, Qin; Chen, Feng

2010-01-01

Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-l-methionine (AdoMet) to 4-hydroxybenzoatemore » in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.« less

A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil1[C][W][OA

PubMed Central

Berim, Anna; Hyatt, David C.; Gang, David R.

2012-01-01

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 4′- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4′-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation. PMID:22923679

Comparative Distribution and Retention of Arsenic in Arsenic (+3 Oxidation State) Methyltransferase Knockout and Wild Type Mice

EPA Science Inventory

The mouse arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a ~ 43 kDa protein that catalyzes conversion of inorganic arsenic into methylated products. Heterologous expression of AS3MT or its silencing by RNA interference controls arsenic methylation phenotypes...

Betaine:homocysteine methyltransferase--a new assay for the liver enzyme and its absence from human skin fibroblasts and peripheral blood lymphocytes.

PubMed

Wang, J A; Dudman, N P; Lynch, J; Wilcken, D E

1991-12-31

Chronic elevation of plasma homocysteine is associated with increased atherogenesis and thrombosis, and can be lowered by betaine (N,N,N-trimethylglycine) treatment which is thought to stimulate activity of the enzyme betaine:homocysteine methyltransferase. We have developed a new assay for this enzyme, in which the products of the enzyme-catalysed reaction between betaine and homocysteine are oxidised by performic acid before being separated and quantified by amino acid analysis. This assay confirmed that human liver contains abundant betaine:homocysteine methyltransferase (33.4 nmol/h/mg protein at 37 degrees C, pH 7.4). Chicken and lamb livers also contain the enzyme, with respective activities of 50.4 and 6.2 nmol/h/mg protein. However, phytohaemagglutinin-stimulated human peripheral blood lymphocytes and cultured human skin fibroblasts contained no detectable betaine:homocysteine methyltransferase (less than 1.4 nmol/h/mg protein), even after cells were pre-cultured in media designed to stimulate production of the enzyme. The results emphasize the importance of the liver in mediating the lowering of elevated circulating homocysteine by betaine.

Insulin-like peptides and DNA/tRNA methyltransferases are involved in the nutritional regulation of female reproduction in Nilaparvata lugens (Stål).

PubMed

Lu, Kai; Chen, Xia; Li, Wenru; Li, Yue; Zhang, Zhichao; Zhou, Qiang

2018-01-10

Insulin-like peptides (ILPs) sense and transduce nutritional information and are linked to female reproduction in many insect species. Our previous studies have shown that "Target of rapamycin" (TOR) pathway functions through juvenile hormone (JH) to regulate amino acids-mediated vitellogenesis in the brown planthopper, Nilaparvata lugens, one of the most destructive rice pests in Asia. Recent reports have demonstrated that DNA methyltransferases (Dnmts) are also involved in female reproduction of N. lugens. However, the roles of ILPs and Dnmts in the nutritional regulation of female reproduction have not been fully elucidated. ILPs and Dnmts are highly expressed in the adult females after a supplement of amino acids, indicating nutrition-stimulated expression patterns of these genes. RNA interference-mediated depletion of NlILP2 or NlILP4 dramatically decreased the expression levels of NlDnmt1 and NlDnmt2 (tRNA methyltransferase), and resulted in severely impaired ovary growth as well as the substantial reduction of fecundity. Notably, NlILP2 or NlILP4 knockdown led to reduced mRNA accumulation of S6 kinase (S6K), a downstream target of the nutritional TOR pathway, and decreased vitellogenin content in the fat body. Silencing NlDnmt1 or NlDnmt2 effectively suppressed ovary development and decreased female fecundity. However, NlDnmt1 or NlDnmt2 knockdown did not influence the expression of NlILP2 and NlILP4. We infer that amino acids act on ILPs and Dnmts to regulate vitellogenesis and oocyte maturation in N. lugens. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

PubMed

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-04-01

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

PubMed Central

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-01-01

Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

Moderation of the effect of adolescent-onset cannabis use on adult psychosis by a functional polymorphism in the catechol-O-methyltransferase gene: longitudinal evidence of a gene X environment interaction.

PubMed

Caspi, Avshalom; Moffitt, Terrie E; Cannon, Mary; McClay, Joseph; Murray, Robin; Harrington, HonaLee; Taylor, Alan; Arseneault, Louise; Williams, Ben; Braithwaite, Antony; Poulton, Richie; Craig, Ian W

2005-05-15

Recent evidence documents that cannabis use by young people is a modest statistical risk factor for psychotic symptoms in adulthood, such as hallucinations and delusions, as well as clinically significant schizophrenia. The vast majority of cannabis users do not develop psychosis, however, prompting us to hypothesize that some people are genetically vulnerable to the deleterious effects of cannabis. In a longitudinal study of a representative birth cohort followed to adulthood, we tested why cannabis use is associated with the emergence of psychosis in a minority of users, but not in others. A functional polymorphism in the catechol-O-methyltransferase (COMT) gene moderated the influence of adolescent cannabis use on developing adult psychosis. Carriers of the COMT valine158 allele were most likely to exhibit psychotic symptoms and to develop schizophreniform disorder if they used cannabis. Cannabis use had no such adverse influence on individuals with two copies of the methionine allele. These findings provide evidence of a gene x environment interaction and suggest that a role of some susceptibility genes is to influence vulnerability to environmental pathogens.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

PubMed Central

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

2011-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

Developmental and Thyroid Hormone Regulation of the DNA Methyltransferase 3a Gene in Xenopus Tadpoles

PubMed Central

Kyono, Yasuhiro; Sachs, Laurent M.; Bilesimo, Patrice; Wen, Luan

2016-01-01

Thyroid hormone is essential for normal development in vertebrates. In amphibians, T3 controls metamorphosis by inducing tissue-specific gene regulation programs. A hallmark of T3 action is the modification of chromatin structure, which underlies changes in gene transcription. We found that mRNA for the de novo DNA methyltransferase (DNMT) dnmt3a, but not dnmt1, increased in the brain of Xenopus tadpoles during metamorphosis in parallel with plasma [T3]. Addition of T3 to the rearing water caused a time-dependent increase in dnmt3a mRNA in tadpole brain, tail, and hind limb. By analyzing data from a genome-wide analysis of T3 receptor (TR) binding in tadpole tail, we identified several putative T3 response elements (TREs) within the dnmt3a locus. Using in vitro DNA binding, transient transfection-reporter, and chromatin immunoprecipitation assays for TRs, we identified two functional TREs at −7.1 kb and +5.1 kb relative to the dnmt3a transcription start site. Sequence alignment showed that these TREs are conserved between two related frog species, X. laevis and X. tropicalis, but not with amniotes. Our previous findings showed that this gene is directly regulated by liganded TRs in mouse brain, and whereas the two mouse TREs are conserved among Eutherian mammals, they are not conserved in Xenopus species. Thus, although T3 regulation of dnmt3a may be an ancient pathway in vertebrates, the genomic sites responsible for hormone regulation may have diverged or arisen by convergent evolution. We hypothesize that direct T3 regulation of dnmt3a may be an important mechanism for modulating global changes in DNA methylation. PMID:27779916

Phenolic profiling of caffeic acid O-methyltransferase-deficient poplar reveals novel benzodioxane oligolignols.

PubMed

Morreel, Kris; Ralph, John; Lu, Fachuang; Goeminne, Geert; Busson, Roger; Herdewijn, Piet; Goeman, Jan L; Van der Eycken, Johan; Boerjan, Wout; Messens, Eric

2004-12-01

Caffeic acid O-methyltransferase (COMT) catalyzes preferentially the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde in monolignol biosynthesis. Here, we have compared HPLC profiles of the methanol-soluble phenolics fraction of xylem tissue from COMT-deficient and control poplars (Populus spp.), using statistical analysis of the peak heights. COMT down-regulation results in significant concentration differences for 25 of the 91 analyzed peaks. Eight peaks were exclusively detected in COMT-deficient poplar, of which four could be purified for further identification using mass spectrometry/mass spectrometry, nuclear magnetic resonance, and spiking of synthesized reference compounds. These new compounds were derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde and were characterized by benzodioxane moieties, a structural type that is also increased in the lignins of COMT-deficient plants. One of these four benzodioxanes amounted to the most abundant oligolignol in the HPLC profile. Furthermore, all of the differentially accumulating oligolignols involving sinapyl units were either reduced in abundance or undetectable. The concentration levels of all identified oligolignols were in agreement with the relative supply of monolignols and with their chemical coupling propensities, which supports the random coupling hypothesis. Chiral HPLC analysis of the most abundant benzodioxane dimer revealed the presence of both enantiomers in equal amounts, indicating that they were formed by radical coupling reactions under simple chemical control rather than guided by dirigent proteins.

Volatile arsenic species released from Escherichia coli expressing the AsIII S-adenosylmethionine methyltransferase gene.

PubMed

Yuan, Chungang; Lu, Xiufen; Qin, Jie; Rosen, Barry P; Le, X Chris

2008-05-01

Biological systems, ranging from bacteria and fungi to humans, can methylate arsenic. Recent studies have suggested that the AsIII S-adenosylmethionine methyltransferase (arsM) gene in bacteria was responsible for the removal of arsenic as the volatile arsines from the bacteria. However, there has been no direct measure of the arsines released from bacteria cultures. We describe here an integrated system incorporating the bacterial incubation and volatile arsenic species analysis, and we demonstrate its application to the identification of the volatile arsines produced in bacterial cultures. The headspace of the bacterial cultures was purged with helium, and the volatile arsenic species were trapped in a chromatographic column immersed in liquid nitrogen. The cryogenically trapped arsines [AsH3, (CH3)AsH2, (CH3)2AsH, and (CH3)3As] were separated by gas chromatography and were detected by inductively coupled plasma mass spectrometry. A hydride generation system was coupled to the bacterial culture system, allowing for spiking standards and for generating calibration arsines necessary for quantitative analysis. Both bacteria containing the arsM gene or its variant arsMC2 gene were able to produce 400-500 ng of trimethylarsine. No trimethylarsine was detectable in bacteria lacking the arsM gene (containing the vector plasmid as negative control). These results confirm that arsM is responsible for releasing arsenic as volatile species from the arsenic-resistant bacteria. Our results also show traces of AsH3, CH3AsH2, and (CH3)2AsH in cultures of bacteria expressing arsM. The method detection limits for AsH3, CH3AsH2, (CH3)2AsH, and (CH3)3As were 0.5, 0.5, 0.7, and 0.6 pg, respectively. The ability to quantify trace levels of these volatile arsenic species makes it possible to study the biotransformation and biochemical roles of the evolution of these volatile arsenic species by biological systems.

Volatile Arsenic Species Released from Escherichia coli Expressing the AsIII S-adenosylmethionine Methyltransferase Gene

PubMed Central

YUAN, CHUNGANG; LU, XIUFEN; QIN, JIE; ROSEN, BARRY P.; LE, X. CHRIS

2015-01-01

Biological systems, ranging from bacteria and fungi to humans, can methylate arsenic. Recent studies have suggested that the AsIII S-adenosylmethionine methyltransferase (arsM) gene in bacteria was responsible for the removal of arsenic as the volatile arsines from the bacteria. However, there has been no direct measure of the arsines released from bacteria cultures. We describe here an integrated system incorporating the bacterial incubation and volatile arsenic species analysis, and we demonstrate its application to the identification of the volatile arsines produced in bacterial cultures. The headspace of the bacterial cultures was purged with helium, and the volatile arsenic species were trapped in a chromatographic column immersed in liquid nitrogen. The cryogenically trapped arsines [AsH3, (CH3)AsH2, (CH3)2AsH, and (CH3)3As] were separated by gas chromatography and were detected by inductively coupled plasma mass spectrometry. A hydride generation system was coupled to the bacterial culture system, allowing for spiking standards and for generating calibration arsines necessary for quantitative analysis. Both bacteria containing the arsM gene or its variant arsMC2 gene were able to produce 400–500 ng of trimethylarsine. No trimethylarsine was detectable in bacteria lacking the arsM gene (containing the vector plasmid as negative control). These results confirm that arsM is responsible for releasing arsenic as volatile species from the arsenic-resistant bacteria. Our results also show traces of AsH3, CH3AsH2, and (CH3)2AsH in cultures of bacteria expressing arsM. The method detection limits for AsH3, CH3AsH2, (CH3)2AsH, and (CH3)3As were 0.5, 0.5, 0.7, and 0.6 pg, respectively. The ability to quantify trace levels of these volatile arsenic species makes it possible to study the biotransformation and biochemical roles of the evolution of these volatile arsenic species by biological systems. PMID:18522094

A Sensitive Luminescent Assay for the Histone Methyltransferase NSD1 and Other SAM-Dependent Enzymes

PubMed Central

Drake, Katherine M.; Watson, Venita G.; Kisielewski, Anne; Glynn, Rebecca

2014-01-01

Abstract A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias. PMID:24927133

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations.

PubMed

Green, Abigail R; Lewis, Kevin M; Barr, John T; Jones, Jeffrey P; Lu, Fachuang; Ralph, John; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

2014-08-01

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low K m for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. © 2014 American Society of Plant Biologists. All Rights Reserved.

MicroRNA396a-5p and -3p induce tomato disease susceptibility by suppressing target genes and upregulating salicylic acid.

PubMed

Chen, Lei; Meng, Jun; Zhai, Junmiao; Xu, Pinsan; Luan, Yushi

2017-12-01

Plants have evolved a variety of mechanisms to perceive and resist the assault of pathogens. The biotrophs, necrotrophs and hemibiotrophs are types of plant pathogens that activate diverse salicylic acid (SA) and jasmonic acid (JA) signaling pathways. In this study we showed that the expressions of miR396a-5p and -3p in Solanum lycopersicum (S. lycopersicum) were both down-regulated after infection by hemibiotroph Phytophthora infestans (P. infestans) and necrotroph Botrytis cinerea (B. cinerea) infection. Overexpression of miR396a-5p and -3p in transgenic tomato enhanced the susceptibility of S. lycopersicum to P. infestans and B. cinerea infection and the tendency to produce reactive oxygen species (ROS) under pathogen-related biotic stress. Additionally, miR396a regulated growth-regulating factor1 (GRF1), salicylic acid carboxyl methyltransferase (SAMT), glycosyl hydrolases (GH) and nucleotide-binding site-leucine-rich repeat (NBS-LRR) and down-regulated their levels. This ultimately led to inhibition of the expression of pathogenesis-related 1 (PR1), TGA transcription factors1 and 2 (TGA1 and TGA2) and JA-dependent proteinase inhibitors I and II (PI I and II), but enhanced the endogenous SA content and nonexpressor of pathogenesis-related genes 1 (NPR1) expression. Taken together, our results showed that negative regulation of target genes and their downstream genes expressions by miR396a-5p and -3p are critical for tomato abiotic stresses via affecting SA or JA signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibitors of Protein Methyltransferases and Demethylases

PubMed Central

2017-01-01

Post-translational modifications of histones by protein methyltransferases (PMTs) and histone demethylases (KDMs) play an important role in the regulation of gene expression and transcription and are implicated in cancer and many other diseases. Many of these enzymes also target various nonhistone proteins impacting numerous crucial biological pathways. Given their key biological functions and implications in human diseases, there has been a growing interest in assessing these enzymes as potential therapeutic targets. Consequently, discovering and developing inhibitors of these enzymes has become a very active and fast-growing research area over the past decade. In this review, we cover the discovery, characterization, and biological application of inhibitors of PMTs and KDMs with emphasis on key advancements in the field. We also discuss challenges, opportunities, and future directions in this emerging, exciting research field. PMID:28338320

Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

PubMed

Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

2014-02-01

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement.

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

PubMed Central

McClelland, M; Nelson, M; Raschke, E

1994-01-01

Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia.

PubMed

Tagde, Ashujit; Rajabi, Hasan; Stroopinsky, Dina; Gali, Reddy; Alam, Maroof; Bouillez, Audrey; Kharbanda, Surender; Stone, Richard; Avigan, David; Kufe, Donald

2016-06-28

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38- population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

Transcriptional regulation by the Set7 lysine methyltransferase

PubMed Central

Keating, Samuel; El-Osta, Assam

2013-01-01

Posttranslational histone modifications define chromatin structure and function. In recent years, a number of studies have characterized many of the enzymatic activities and diverse regulatory components required for monomethylation of histone H3 lysine 4 (H3K4me1) and the expression of specific genes. The challenge now is to understand how this specific chemical modification is written and the Set7 methyltransferase has emerged as a key regulatory enzyme mediating methylation of lysine residues of histone and non-histone proteins. In this review, we comprehensively explore the regulatory proteins modified by Set7 and highlight mechanisms of specific co-recruitment of the enzyme to activating promoters. With a focus on signaling and transcriptional control in disease we discuss recent experimental data emphasizing specific components of diverse regulatory complexes that mediate chromatin modification and reinterpretation of Set7-mediated gene expression. PMID:23478572

The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

PubMed

Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle

2005-10-18

We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

Histone H3 Lysine 36 Methyltransferase Whsc1 Promotes the Association of Runx2 and p300 in the Activation of Bone-Related Genes

PubMed Central

Lee, Yu Fei; Nimura, Keisuke; Lo, Wan Ning; Saga, Kotaro; Kaneda, Yasufumi

2014-01-01

The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1 −/− embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression. PMID:25188294

PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine.

PubMed

Lee, Jin-Hyung; Cook, Jeffry R; Yang, Zhi-Hong; Mirochnitchenko, Olga; Gunderson, Samuel I; Felix, Arthur M; Herth, Nicole; Hoffmann, Ralf; Pestka, Sidney

2005-02-04

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

COBALAMIN- AND COBAMIDE-DEPENDENT METHYLTRANSFERASES

PubMed Central

Matthews, Rowena G.; Koutmos, Markos; Datta, Supratim

2008-01-01

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple SN2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co+1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled. PMID:19059104

Maize OXIDATIVE STRESS2 Homologs Enhance Cadmium Tolerance in Arabidopsis through Activation of a Putative SAM-Dependent Methyltransferase Gene.

PubMed

He, Lilong; Ma, Xiaoling; Li, Zhenzhen; Jiao, Zhengli; Li, Yongqing; Ow, David W

2016-07-01

Previously the Arabidopsis (Arabidopsis thaliana) zinc finger protein OXIDATIVE STRESS2 (AtOXS2) and four OXS2-like (AtO2L) family members were described to play a role in stress tolerance and stress escape. For stress escape, SOC1 was a target of AtOXS2. However, for stress tolerance, the downstream targets were not identified. We cloned two OXS2 homolog genes from sweet corn, ZmOXS2b and ZmO2L1 Both genes are transiently inducible by Cd treatment. When expressed in Arabidopsis, each enhances tolerance against cadmium. Further analysis showed that ZmOXS2b and ZmO2L1 proteins enhance Cd tolerance in Arabidopsis by activating at least one target gene, that encoding a putative S-adenosyl-l-Met-dependent methyltransferase superfamily protein (AT5G37990), which we named CIMT1 This activation involves the in vivo interaction with a segment of the CIMT1 promoter that contains a BOXS2 motif previously identified as the binding element for AtOXS2. More importantly, CIMT1 is induced by Cd treatment, and overexpression of this gene alone was sufficient to enhance Cd tolerance in Arabidopsis. The connection of ZmOXS2b and ZmO2L1 to Arabidopsis CIMT1 suggests a similar network may exist in maize (Zea mays) and may provide a clue to possibly using a CIMT1 maize homolog to engineer stress tolerance in a major crop. © 2016 American Society of Plant Biologists. All Rights Reserved.

Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.

PubMed

Atta, Hussein; El-Rehany, Mahmoud; Hammam, Olfat; Abdel-Ghany, Hend; Ramzy, Maggie; Roderfeld, Martin; Roeb, Elke; Al-Hendy, Ayman; Raheim, Salama Abdel; Allam, Hatem; Marey, Heba

2014-01-01

Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Cloning, expression, purification and preliminary X-­ray crystallographic analysis of mouse protein arginine methyltransferase 7

PubMed Central

Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. PMID:24419624

Phenolic Profiling of Caffeic Acid O-Methyltransferase-Deficient Poplar Reveals Novel Benzodioxane Oligolignols1

PubMed Central

Morreel, Kris; Ralph, John; Lu, Fachuang; Goeminne, Geert; Busson, Roger; Herdewijn, Piet; Goeman, Jan L.; Van der Eycken, Johan; Boerjan, Wout; Messens, Eric

2004-01-01

Caffeic acid O-methyltransferase (COMT) catalyzes preferentially the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde in monolignol biosynthesis. Here, we have compared HPLC profiles of the methanol-soluble phenolics fraction of xylem tissue from COMT-deficient and control poplars (Populus spp.), using statistical analysis of the peak heights. COMT down-regulation results in significant concentration differences for 25 of the 91 analyzed peaks. Eight peaks were exclusively detected in COMT-deficient poplar, of which four could be purified for further identification using mass spectrometry/mass spectrometry, nuclear magnetic resonance, and spiking of synthesized reference compounds. These new compounds were derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde and were characterized by benzodioxane moieties, a structural type that is also increased in the lignins of COMT-deficient plants. One of these four benzodioxanes amounted to the most abundant oligolignol in the HPLC profile. Furthermore, all of the differentially accumulating oligolignols involving sinapyl units were either reduced in abundance or undetectable. The concentration levels of all identified oligolignols were in agreement with the relative supply of monolignols and with their chemical coupling propensities, which supports the random coupling hypothesis. Chiral HPLC analysis of the most abundant benzodioxane dimer revealed the presence of both enantiomers in equal amounts, indicating that they were formed by radical coupling reactions under simple chemical control rather than guided by dirigent proteins. PMID:15563622

An intact SAM-dependent methyltransferase fold is encoded by the human endothelin-converting enzyme-2 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tempel, W.; Wu, H.; Dombrovsky, L.

2010-08-17

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment andmore » a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptide with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

PubMed Central

Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J. M.; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D. James; Carter, Melissa T.; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B.

2015-01-01

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. PMID:26206890

Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

PubMed Central

Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

2017-01-01

The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This

Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6more » but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.« less

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

The AMT1 Arginine Methyltransferase Gene Is Important for Plant Infection and Normal Hyphal Growth in Fusarium graminearum

PubMed Central

Hou, Rui; Zhou, Xiaoying; Li, Guotian; Zhang, Shijie; Xu, Jin-Rong

2012-01-01

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection. PMID:22693618

Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop.

PubMed

Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G

2014-11-21

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations1[W

PubMed Central

Green, Abigail R.; Lewis, Kevin M.; Barr, John T.; Jones, Jeffrey P.; Lu, Fachuang; Ralph, John; Vermerris, Wilfred; Sattler, Scott E.; Kang, ChulHee

2014-01-01

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. PMID:24948836

Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study.

PubMed

Fonteneau, M; Filliol, D; Anglard, P; Befort, K; Romieu, P; Zwiller, J

2017-03-01

DNA methylation is a major epigenetic process which regulates the accessibility of genes to the transcriptional machinery. In the present study, we investigated whether modifying the global DNA methylation pattern in the brain would alter cocaine intake by rats, using the cocaine self-administration test. The data indicate that treatment of rats with the DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (dAZA) and zebularine enhanced the reinforcing properties of cocaine. To obtain some insights about the underlying neurobiological mechanisms, a genome-wide methylation analysis was undertaken in the prefrontal cortex of rats self-administering cocaine and treated with or without dAZA. The study identified nearly 189 000 differentially methylated regions (DMRs), about half of them were located inside gene bodies, while only 9% of DMRs were found in the promoter regions of genes. About 99% of methylation changes occurred outside CpG islands. Gene expression studies confirmed the inverse correlation usually observed between increased methylation and transcriptional activation when methylation occurs in the gene promoter. This inverse correlation was not observed when methylation took place inside gene bodies. Using the literature-based Ingenuity Pathway Analysis, we explored how the differentially methylated genes were related. The analysis showed that increase in cocaine intake by rats in response to DNA methyltransferase inhibitors underlies plasticity mechanisms which mainly concern axonal growth and synaptogenesis as well as spine remodeling. Together with the Akt/PI3K pathway, the Rho-GTPase family was found to be involved in the plasticity underlying the effect of dAZA on the observed behavioral changes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

PubMed Central

Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

2016-01-01

Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

Simultaneous Down-Regulation of Caffeic/5-Hydroxy Ferulic Acid-O-Methyltransferase I and Cinnamoyl-Coenzyme A Reductase in the Progeny from a Cross between Tobacco Lines Homozygous for Each Transgene. Consequences for Plant Development and Lignin Synthesis1

PubMed Central

Pinçon, Gaelle; Chabannes, Matthieu; Lapierre, Catherine; Pollet, Brigitte; Ruel, Katia; Joseleau, Jean-Paul; Boudet, Alain M.; Legrand, Michel

2001-01-01

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

PubMed

Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

2014-05-06

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

PubMed

Gou, Qing; He, ShuJiao; Zhou, ZeJian

2017-02-01

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

PubMed

Stepper, Peter; Kungulovski, Goran; Jurkowska, Renata Z; Chandra, Tamir; Krueger, Felix; Reinhardt, Richard; Reik, Wolf; Jeltsch, Albert; Jurkowski, Tomasz P

2017-02-28

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The inhibition of the mammalian DNA methyltransferase 3a (Dnmt3a) by dietary black tea and coffee polyphenols

PubMed Central

2011-01-01

Background Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean. Results We studied the inhibition of DNA methyltransferase 3a by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance. Conclusions Since Dnnmt3a has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols. PMID:21510884

Development of fluorescent methods for DNA methyltransferase assay

NASA Astrophysics Data System (ADS)

Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

Gene quantification by the NanoGene assay is resistant to inhibition by humic acids.

PubMed

Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong

2011-10-15

NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (<100 μg/mL). However, a substantial amount of nonspecific binding was observed between the humic acids and target gDNA. This possibly results in lesser amount of target gDNA being captured by the probe and signaling DNA.

Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

PubMed Central

Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

2016-01-01

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome

PubMed Central

Kim, Yuna; Lee, Hyeong-Min; Xiong, Yan; Sciaky, Noah; Hulbert, Samuel W; Cao, Xinyu; Everitt, Jeffrey I; Jin, Jian; Roth, Bryan L; Jiang, Yong-hui

2017-01-01

Prader–Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11–q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642—two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)—activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS−U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS−U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS. PMID:28024084

DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

PubMed

Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

2016-10-12

DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

DOEpatents

Liu, Chang-Jun; Cai, Yuanheng

2017-01-31

Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

Human Calmodulin Methyltransferase: Expression, Activity on Calmodulin, and Hsp90 Dependence

PubMed Central

Magen, Sophia; Magnani, Roberta; Haziza, Sitvanit; Hershkovitz, Eli; Houtz, Robert; Cambi, Franca; Parvari, Ruti

2012-01-01

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional transcripts in cells of the 2p21 deletion syndrome patients that start from alternative exons positioned outside the deletion region. One of them starts in the 2nd known exon, the other in a novel exon. The transcript starting from the novel exon was also identified in a variety of tissues from normal individuals. These new transcripts are not expected to produce proteins. Immunofluorescent localization of tagged CaM KMT in HeLa cells indicates that it is present in both the cytoplasm and nucleus of cells whereas the short isoform is localized to the Golgi apparatus. Using Western blot analysis we show that the CaM KMT protein is broadly expressed in mouse tissues. Finally we demonstrate that the CaM KMT interacts with the middle portion of the Hsp90 molecular chaperon and is probably a client protein since it is degraded upon treatment of cells with the Hsp90 inhibitor geldanamycin. These findings suggest that the CaM KMT is the major, possibly the single, methyltransferase of calmodulin in human cells with a wide tissue distribution and is a novel Hsp90 client protein. Thus our data provides basic information for a gene potentially contributing to the patient phenotype of two contiguous gene deletion syndromes. PMID:23285036

Human calmodulin methyltransferase: expression, activity on calmodulin, and Hsp90 dependence.

PubMed

Magen, Sophia; Magnani, Roberta; Haziza, Sitvanit; Hershkovitz, Eli; Houtz, Robert; Cambi, Franca; Parvari, Ruti

2012-01-01

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional transcripts in cells of the 2p21 deletion syndrome patients that start from alternative exons positioned outside the deletion region. One of them starts in the 2(nd) known exon, the other in a novel exon. The transcript starting from the novel exon was also identified in a variety of tissues from normal individuals. These new transcripts are not expected to produce proteins. Immunofluorescent localization of tagged CaM KMT in HeLa cells indicates that it is present in both the cytoplasm and nucleus of cells whereas the short isoform is localized to the Golgi apparatus. Using Western blot analysis we show that the CaM KMT protein is broadly expressed in mouse tissues. Finally we demonstrate that the CaM KMT interacts with the middle portion of the Hsp90 molecular chaperon and is probably a client protein since it is degraded upon treatment of cells with the Hsp90 inhibitor geldanamycin. These findings suggest that the CaM KMT is the major, possibly the single, methyltransferase of calmodulin in human cells with a wide tissue distribution and is a novel Hsp90 client protein. Thus our data provides basic information for a gene potentially contributing to the patient phenotype of two contiguous gene deletion syndromes.

Challenges in profiling and lead optimization of drug discovery for methyltransferases.

PubMed

Horiuchi, Kurumi Y

2015-11-01

The importance of epigenetics in the initiation and progression of disease has attracted many investigators to incorporate this novel and exciting field in drug development. Protein methyltransferases are one of the target classes which have gained attention as potential therapeutic targets after promising results of inhibitors for EZH2 and DOT1L in clinical trials. There are many technologies developed in order to find small molecule inhibitors for protein methyltransferases. However, in contrast to high throughput screening, profiling against different methyltransferases is challenging since each enzyme has a different substrate preference so that it is hard to profile in one assay format. Here, different technologies for methyltransferase assays will be overviewed, and the advantages and disadvantages of each will be discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quercetin-Induced Lifespan Extension in Podospora anserina Requires Methylation of the Flavonoid by the O-Methyltransferase PaMTH1.

PubMed

Warnsmann, Verena; Hainbuch, Saskia; Osiewacz, Heinz D

2018-01-01

Quercetin is a flavonoid that is ubiquitously found in vegetables and fruits. Like other flavonoids, it is active in balancing cellular reactive oxygen species (ROS) levels and has a cyto-protective function. Previously, a link between ROS balancing, aging, and the activity of O -methyltransferases was reported in different organisms including the aging model Podospora anserina. Here we describe a role of the S -adenosylmethionine-dependent O -methyltransferase PaMTH1 in quercetin-induced lifespan extension. We found that effects of quercetin treatment depend on the methylation state of the flavonoid. Specifically, we observed that quercetin treatment increases the lifespan of the wild type but not of the PaMth1 deletion mutant. The lifespan increasing effect is not associated with effects of quercetin on mitochondrial respiration or ROS levels but linked to the induction of the PaMth1 gene. Overall, our data demonstrate a novel role of O -methyltransferase in quercetin-induced longevity and identify the underlying pathway as part of a network of longevity assurance pathways with the perspective to intervene into mechanisms of biological aging.

[Relationship between single nucleotide polymorphisms in thiopurine methyltransferase gene and tolerance to thiopurines in acute leukemia].

PubMed

Ma, Xiao-li; Zhu, Ping; Wu, Min-yuan; Li, Zhi-gang; Hu, Ya-mei

2003-12-01

For the purpose of clarifying the influence of thiopurine methyltransferase (TPMT) gene single nucleotide polymorphisms (SNPs) on the efficacy of thiopurines and risk for its toxicity and therefore improving the safety and efficacy of thiopurines, the authors investigated TPMT genotype in acute leukemia in children who were intolerant to the treatment with 6-mercap topurine (6-MP). TPMT genotype was determined in an unrelated population of 250 Chinese healthy blood donors and 280 children with acute leukemia. TPMT genotyping assay was based on polymerase chain reaction (PCR), restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing in the TPMT * 2 (G238C), TPMT * 3A (G460A, A719G) and TPMT * 3C (A719G). There were 10 TPMT * 1/TPMT * 3C heterozygotes in 280 children. The frequency of the polymorphism was 3.6%. All the involved alleles were TPMT * 3C. Of the 160 children acute leukemia evaluated, 45 (26%) were intolerant to 6-MP. Presentations included hepatotoxicity and hematological toxicity. Six out of 45 children were heterozygous, while the other 39 were wild type homozygous. Before dosage adjustments for thiopurine, the hematologic toxicity and hepatotoxicity in TPMT heterozygous individuals occurred more frequently than in homozygous. Therefore, cases of TPMT heterozygotes experienced more missed doses of 6-MP. TPMT genotype is associated with tolerance in acute leukemia in children. The heterozygote individuals have low TPMT activity. Therefore the frequencies of hemtopoietic toxicity and hepatoxicity are high after using 6-MP. Detection of SNPs in the TPMT genes is useful in identifying children before administration of 6-MP.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

PubMed

Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J M; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D James; Carter, Melissa T; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B

2015-10-15

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Catechol-O-Methyltransferase "Val[superscript 158]Met" Genotype, Parenting Practices and Adolescent Alcohol Use: Testing the Differential Susceptibility Hypothesis

ERIC Educational Resources Information Center

Laucht, Manfred; Blomeyer, Dorothea; Buchmann, Arlette F.; Treutlein, Jens; Schmidt, Martin H.; Esser, Gunter; Jennen-Steinmetz, Christine; Rietschel, Marcella; Zimmermann, Ulrich S.; Banaschewski, Tobias

2012-01-01

Background: Recently, first evidence has been reported for a gene-parenting interaction (G x E) with regard to adolescent alcohol use. The present investigation set out to extend this research using the catechol-O-methyltransferase ("COMT") "Val[superscript 158]Met" polymorphism as a genetic susceptibility factor. Moreover, the current study…

Structure and function of flavivirus NS5 methyltransferase.

PubMed

Zhou, Yangsheng; Ray, Debashish; Zhao, Yiwei; Dong, Hongping; Ren, Suping; Li, Zhong; Guo, Yi; Bernard, Kristen A; Shi, Pei-Yong; Li, Hongmin

2007-04-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

Structure and Function of Flavivirus NS5 Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou,Y.; Ray, D.; Zhao, Y.

2007-01-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showedmore » a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.« less

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-05-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.

Hexanoic acid is a resistance inducer that protects tomato plants against Pseudomonas syringae by priming the jasmonic acid and salicylic acid pathways.

PubMed

Scalschi, Loredana; Vicedo, Begonya; Camañes, Gemma; Fernandez-Crespo, Emma; Lapeña, Leonor; González-Bosch, Carmen; García-Agustín, Pilar

2013-05-01

Hexanoic acid-induced resistance (Hx-IR) is effective against several pathogens in tomato plants. Our study of the mechanisms implicated in Hx-IR against Pseudomonas syringae pv. tomato DC3000 suggests that hexanoic acid (Hx) treatment counteracts the negative effect of coronatine (COR) and jasmonyl-isoleucine (JA-Ile) on the salicylic acid (SA) pathway. In Hx-treated plants, an increase in the expression of jasmonic acid carboxyl methyltransferase (JMT) and the SA marker genes PR1 and PR5 indicates a boost in this signalling pathway at the expense of a decrease in JA-Ile. Moreover, Hx treatment potentiates 12-oxo-phytodienoic acid accumulation, which suggests that this molecule might play a role per se in Hx-IR. These results support a positive relationship between the SA and JA pathways in Hx-primed plants. Furthermore, one of the mechanisms of virulence mediated by COR is stomatal re-opening on infection with P. syringae. In this work, we observed that Hx seems to inhibit stomatal opening in planta in the presence of COR, which suggests that, on infection in tomato, this treatment suppresses effector action to prevent bacterial entry into the mesophyll. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference.

PubMed

Louie, Gordon V; Bowman, Marianne E; Tu, Yi; Mouradov, Aidyn; Spangenberg, German; Noel, Joseph P

2010-12-01

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.

Inactivation of SAM-methyltransferase is the mechanism of attenuation of a historic louse borne typhus vaccine strain.

PubMed

Liu, Yan; Wu, Bin; Weinstock, George; Walker, David H; Yu, Xue-Jie

2014-01-01

Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

USDA-ARS?s Scientific Manuscript database

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

PubMed

Matsuoka, Taeko; Kawai, Koji; Ando, Satoshi; Sugita, Shintaro; Kandori, Shuya; Kojima, Takahiro; Miyazaki, Jun; Nishiyama, Hiroyuki

2016-05-01

DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

NASA Astrophysics Data System (ADS)

Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

2016-07-01

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Mouse Vk gene classification by nucleic acid sequence similarity.

PubMed

Strohal, R; Helmberg, A; Kroemer, G; Kofler, R

1989-01-01

Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.

The fish pathogen Yersinia ruckeri produces holomycin and uses an RNA methyltransferase for self-resistance.

PubMed

Qin, Zhiwei; Baker, Alexander Thomas; Raab, Andrea; Huang, Sheng; Wang, Tiehui; Yu, Yi; Jaspars, Marcel; Secombes, Christopher J; Deng, Hai

2013-05-24

Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

PubMed Central

Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David

2017-01-01

Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623

Aurora-B Regulates RNA Methyltransferase NSUN2

PubMed Central

Sakita-Suto, Shiho; Kanda, Akifumi; Suzuki, Fumio; Sato, Sunao; Takata, Takashi

2007-01-01

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G1 phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis. PMID:17215513

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.

PubMed

Panikar, Chitra S; Rajpathak, Shriram N; Abhyankar, Varada; Deshmukh, Saniya; Deobagkar, Deepti D

2015-12-01

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

Analysis of the Association between Catechol-O-Methyltransferase Val158Met and Male Sexual Orientation.

PubMed

Yu, Wei; Tu, Dan; Hong, Fuchang; Wang, Jing; Liu, Xiaoli; Cai, Yumao; Xu, Ruiwei; Zhao, Guanglu; Wang, Feng; Pan, Hong; Wu, Shinan; Feng, Tiejian; Wang, Binbin

2015-09-01

Male sexual orientation is thought to have a genetic component. However, previous studies have failed to generate positive results from among candidate genes. Catechol-O-methyltransferase (COMT), located on chromosome 22, has six exons, spans 27 kb, and encodes a protein of 271 amino acids. COMT has an important role in regulating the embryonic levels of catecholamine neurotransmitters (such as dopamine, norepinephrine, and epinephrine) and estrogens. COMT is also thought to be related to sexual orientation. This study aimed to investigate the relationship between the COMT Val158Met variant and male sexual orientation. We performed association analysis of the COMT gene single nucleotide polymorphism, Val158Met, in 409 homosexual cases and 387 heterosexual control Chinese men. COMT polymorphism status was determined using a polymerase chain reaction-based assay. Polymerase chain reaction was performed to genotype the COMT Val158Met polymorphism. The frequency differences of the genotype and alleles distribution between the male homosexual and control groups. Significant differences, both in genotype and alleles, between male homosexual individuals and controls indicated a genetic component related to male homosexuality. The Val allele recessive model could be an interrelated genetic model of the cause of male homosexuality. The COMT Val158Met variant might be associated with male sexual orientation and a recessive model was suggested. © 2015 International Society for Sexual Medicine.

Involvement of human histamine N-methyltransferase gene polymorphisms in susceptibility to atopic dermatitis in korean children.

PubMed

Lee, Hee Seon; Kim, Seung-Hyun; Kim, Kyung Won; Baek, Ji Young; Park, Hae-Sim; Lee, Kyung Eun; Hong, Jung Yeon; Kim, Mi Na; Heo, Won Il; Sohn, Myung Hyun; Kim, Kyu-Earn

2012-01-01

Histamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children. We genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed. Of the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype. Polymorphisms in HNMT appear to confer susceptibility to AD in Korean children.

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases.

PubMed

Yang, Ya-Ling; Wang, Feng-Sheng; Li, Sung-Chou; Tiao, Mao-Meng; Huang, Ying-Hsien

2017-01-18

MicroRNA-29 (miR-29) is found to modulate hepatic stellate cells' (HSCs) activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to bile duct-ligation (BDL) to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A) expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine.

PubMed

Emmert, J L; Garrow, T A; Baker, D H

1996-08-01

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.

Differential effects of thiopurine methyltransferase (TPMT) and multidrug resistance-associated protein gene 4 (MRP4) on mercaptopurine toxicity.

PubMed

Liu, Chengcheng; Janke, Laura J; Yang, Jun J; Evans, William E; Schuetz, John D; Relling, Mary V

2017-08-01

Mercaptopurine plays a pivotal role in treatment of acute lymphoblastic leukemia (ALL) and autoimmune diseases, and inter-individual variability in mercaptopurine tolerance can influence treatment outcome. Thiopurine methyltransferase (TPMT) and multi-drug resistant Protein 4 (MRP4) have both been associated with mercaptopurine toxicity in clinical studies, but their relative contributions remain unclear. We studied the metabolism of and tolerance to mercaptopurine in murine knockout models of Tpmt, Mrp4, and both genes simultaneously. Upon mercaptopurine treatment, Tpmt -/- Mrp4 -/- mice had the highest concentration of bone marrow thioguanine nucleotides (8.5 pmol/5 × 10 6 cells, P = 7.8 × 10 -4 compared with 2.7 pmol/5 × 10 6 cells in wild-types), followed by those with Mrp4 or Tpmt deficiency alone (6.1 and 4.3 pmol/5 × 10 6 cells, respectively). Mrp4-deficient mice accumulated higher concentrations of methylmercaptopurine metabolites compared with wild-type (76.5 vs. 23.2 pmol/5 × 10 6 cells, P = 0.027). Mice exposed to a clinically relevant mercaptopurine dosing regimen displayed differences in toxicity and survival among the genotypes. The double knock-out of both genes experienced greater toxicity and shorter survival compared to the single knockout of either Tpmt (P = 1.7 × 10 -6 ) or Mrp4 (P = 7.4 × 10 -10 ). We showed that both Tpmt and Mrp4 influence mercaptopurine disposition and toxicity.

Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain

PubMed Central

Liu, Yan; Wu, Bin; Weinstock, George; Walker, David H.; Yu, Xue-jie

2014-01-01

Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine. PMID:25412248

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

PubMed Central

Zhong, Xuehua; Hale, Christopher J.; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A.; Henderson, Ian R.; Wohlschlegel, James A.; Jacobsen, Steven E.

2015-01-01

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

Role of O-methyltransferase in the lignification of Douglas-fir cultured tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monroe, S.H.

1983-01-01

O-methyltransferase (OMT) is a key enzyme in the biosynthesis of lignin. This enzyme was isolated and characterized in an effort to understand why Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) callus tissue does not form appreciable amounts of lignin yet does form large amounts of the related flavonoids and tannins. It was shown that the OMT in the callus tissue is a cell wall associated, membrane-bound enzyme, in contrast to that of all reported plant species and to Douglas-fir seedlings, which have either a microsomal or soluble OMT. The effect this had on the OMT kinetic constants was studied. It was foundmore » that the callus OMT had much higher K/sub m/ constants for caffeic acid in both the membrane-bound and free forms compared with seedlings. The callus membrane-bound K/sub m/ for caffeic acid is 333 ..mu..M. The callus membrane-free K/sub m/ for caffeic acid is 250 ..mu..M. The seedling K/sub m/ for caffeic acid is 90 ..mu..M.« less

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

PubMed

Pillinger, M H; Volker, C; Stock, J B; Weissmann, G; Philips, M R

1994-01-14

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

DNA methylation of amino acid transporter genes in the human placenta.

PubMed

Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

2017-12-01

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase.

PubMed

Rothe, Grit; Hachiya, Akira; Yamada, Yasuyuki; Hashimoto, Takashi; Dräger, Birgit

2003-09-01

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.

Methylation of Arsenic by Recombinant Human Wild-Type Arsenic (+3 Oxidation State) Methyltransferase and its Methionine 287 Threonine (M287T) Polymorph

EPA Science Inventory

ABSTRACT Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency...

Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

PubMed

Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

2003-01-01

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling

PubMed Central

Pontier, Dominique; Albrieux, Catherine; Joyard, Jacques; Lagrange, Thierry; Block, Maryse

2007-01-01

Protoporphyrin IX is the last common intermediate between the haem and chlorophyll biosynthesis pathways. The addition of Mg directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the Mg chelatase and is a highly regulated process. The corresponding product, Mg protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. In order to get more information on the chlorophyll biosynthesis pathway and on Mg protoporphyrin IX derivative functions, we have identified an Mg protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from Mg protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cyt b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of Mg protoporphyrin IX methylester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the Mg chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step. PMID:17135235

The relative contribution of genes operating in the S-methylmethionine cycle to methionine metabolism in Arabidopsis seeds.

PubMed

Cohen, Hagai; Salmon, Asaf; Tietel, Zipora; Hacham, Yael; Amir, Rachel

2017-05-01

Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine synthesis in seeds. In addition, mutual effects exist between the S -methylmethionine cycle and the aspartate family pathway in seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. The previous lines of evidence proposed that the S-methylmethionine (SMM) cycle contributes to methionine synthesis in seeds where methionine that is produced in non-seed tissues is converted to SMM and then transported via the phloem into the seeds. However, the relative regulatory roles of the S-methyltransferases operating within this cycle in seeds are yet to be fully understood. In the current study, we generated transgenic Arabidopsis seeds with altered expression of three HOMOCYSTEINE S-METHYLTRANSFERASEs (HMTs) and METHIONINE S-METHYLTRANSFERASE (MMT), and profiled them for transcript and metabolic changes. The results revealed that AtHMT1 and AtHMT3, but not AtHMT2 and AtMMT, are the predominant enzymes operating in seeds as altered expression of these two genes affected the levels of methionine and SMM in transgenic seeds. Their manipulations resulted in adapted expression level of genes participating in methionine synthesis through the SMM and aspartate family pathways. Taken together, our findings provide new insights into the regulatory roles of the SMM cycle and the mutual effects existing between the two methionine biosynthesis pathways, highlighting the complexity of the metabolism of methionine and SMM in seeds.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD

PubMed Central

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-01-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosyn­thesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-l-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit. PMID:24817736

Crystal Structure of the Escherichia coli 23S rRNA: m{5}C Methyltransferase RlmI (YccW) Reveals Evolutionary Links Between RNA Modification Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunita, S.; Tkaczuk, K; Purta, E

2008-01-01

Methylation is the most common RNA modification in the three domains of life. Transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to specific atoms of RNA nucleotides is catalyzed by methyltransferase (MTase) enzymes. The rRNA MTase RlmI (rRNA large subunit methyltransferase gene I; previously known as YccW) specifically modifies Escherichia coli 23S rRNA at nucleotide C1962 to form 5-methylcytosine. Here, we report the crystal structure of RlmI refined at 2 {angstrom} to a final R-factor of 0.194 (R{sub free} = 0.242). The RlmI molecule comprises three domains: the N-terminal PUA domain; the central domain, which resembles a domain previously foundmore » in RNA:5-methyluridine MTases; and the C-terminal catalytic domain, which contains the AdoMet-binding site. The central and C-terminal domains are linked by a {Beta}-hairpin structure that has previously been observed in several MTases acting on nucleic acids or proteins. Based on bioinformatics analyses, we propose a model for the RlmI-AdoMet-RNA complex. Comparative structural analyses of RlmI and its homologs provide insight into the potential function of several structures that have been solved by structural genomics groups and furthermore indicate that the evolutionary paths of RNA and DNA 5-methyluridine and 5-methylcytosine MTases have been closely intertwined.« less

Monolignol 4-O-methyltransferases and uses thereof

DOEpatents

Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

2014-11-18

Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil.

PubMed

Pham, Anh-Tung; Shannon, J Grover; Bilyeu, Kristin D

2012-08-01

High oleic acid soybeans were produced by combining mutant FAD2-1A and FAD2-1B genes. Despite having a high oleic acid content, the linolenic acid content of these soybeans was in the range of 4-6 %, which may be high enough to cause oxidative instability of the oil. Therefore, a study was conducted to incorporate one or two mutant FAD3 genes into the high oleic acid background to further reduce the linolenic acid content. As a result, soybean lines with high oleic acid and low linolenic acid (HOLL) content were produced using different sources of mutant FAD2-1A genes. While oleic acid content of these HOLL lines was stable across two testing environments, the reduction of linolenic acid content varied depending on the number of mutant FAD3 genes combined with mutant FAD2-1 genes, on the severity of mutation in the FAD2-1A gene, and on the testing environment. Combination of two mutant FAD2-1 genes and one mutant FAD3 gene resulted in less than 2 % linolenic acid content in Portageville, Missouri (MO) while four mutant genes were needed to achieve the same linolenic acid in Columbia, MO. This study generated non-transgenic soybeans with the highest oleic acid content and lowest linolenic acid content reported to date, offering a unique alternative to produce a fatty acid profile similar to olive oil.

Structure-Function Analyses of a Caffeic Acid O-Methyltransferase from Perennial Ryegrass Reveal the Molecular Basis for Substrate Preference[W][OA

PubMed Central

Louie, Gordon V.; Bowman, Marianne E.; Tu, Yi; Mouradov, Aidyn; Spangenberg, German; Noel, Joseph P.

2010-01-01

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-l-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT’s catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde. PMID:21177481

Catechol-O-methyltransferase (COMT) gene modulates private self-consciousness and self-flexibility.

PubMed

Wang, Bei; Ru, Wenzhao; Yang, Xing; Yang, Lu; Fang, Pengpeng; Zhu, Xu; Shen, Guomin; Gao, Xiaocai; Gong, Pingyuan

2016-08-01

Dopamine levels in the brain influence human consciousness. Inspired by the role of Catechol-O-methyltransferase (COMT) in inactivating dopamine in the brain, we investigated to what extent COMT could modulate individual's self-consciousness dispositions and self-consistency by genotyping the COMT Val158Met (rs4680) polymorphism and measuring self-consciousness and self-consistency and congruence in a college student population. The results indicated that COMT Val158Met polymorphism significantly modulated the private self-consciousness. The individuals with Val/Val genotype, corresponding to lower dopamine levels in the brain, were more likely to be aware of their feelings and beliefs. The results also indicated that this polymorphism modulated one's self-flexibility. The individuals with Val/Val genotype showed higher levels of stereotype in self-concept compared with those with Met/Met genotype. These findings suggest that COMT is a predictor of the individual differences in self-consciousness and self-flexibility. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel BLAST-Based Relative Distance (BBRD) method can effectively group members of protein arginine methyltransferases and suggest their evolutionary relationship.

PubMed

Wang, Yi-Chun; Wang, Jing-Doo; Chen, Chin-Han; Chen, Yi-Wen; Li, Chuan

2015-03-01

We developed a novel BLAST-Based Relative Distance (BBRD) method by Pearson's correlation coefficient to avoid the problems of tedious multiple sequence alignment and complicated outgroup selection. We showed its application on reconstructing reliable phylogeny for nucleotide and protein sequences as exemplified by the fmr-1 gene and dihydrolipoamide dehydrogenase, respectively. We then used BBRD to resolve 124 protein arginine methyltransferases (PRMTs) that are homologues of nine mammalian PRMTs. The tree placed the uncharacterized PRMT9 with PRMT7 in the same clade, outside of all the Type I PRMTs including PRMT1 and its vertebrate paralogue PRMT8, PRMT3, PRMT6, PRMT2 and PRMT4. The PRMT7/9 branch then connects with the type II PRMT5. Some non-vertebrates contain different PRMTs without high sequence homology with the mammalian PRMTs. For example, in the case of Drosophila arginine methyltransferase (DART) and Trypanosoma brucei methyltransferases (TbPRMTs) in the analyses, the BBRD program grouped them with specific clades and thus suggested their evolutionary relationships. The BBRD method thus provided a great tool to construct a reliable tree for members of protein families through evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

TERRA and the histone methyltransferase Dot1 cooperate to regulate senescence in budding yeast

PubMed Central

Wanat, Jennifer J.; Logsdon, Glennis A.; Driskill, Jordan H.; Deng, Zhong; Lieberman, Paul M.

2018-01-01

The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (tlc1Δ). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA in vivo and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of EXO1, TEL1, SAS2, and genes encoding RNase H enzymes. Further, it acts independently of the senescence delay provided by RAD52-dependent recombination. However, anti-TERRA delays senescence in a fashion epistatic to inactivation of the conserved histone methyltransferase Dot1. Dot1 associates with TERRA, and anti-TERRA disrupts this interaction in vitro and in vivo. Surprisingly, the anti-TERRA delay is independent of the C-terminal methyltransferase domain of Dot1 and instead requires only its N-terminus, which was previously found to facilitate release of telomeres from the nuclear periphery. Together, these data suggest that TERRA and Dot1 cooperate to drive senescence. PMID:29649255

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

PubMed

Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

2016-04-04

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals

PubMed Central

Thomas, David J.; Li, Jiaxin; Waters, Stephen B.; Xing, Weibing; Adair, Blakely M.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav

2008-01-01

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme’s role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway. PMID:17202581

Epigenetic Guardian: A Review of the DNA Methyltransferase DNMT3A in Acute Myeloid Leukaemia and Clonal Haematopoiesis.

PubMed

Chaudry, Sabah F; Chevassut, Timothy J T

2017-01-01

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by clonal stem cell proliferation and aberrant block in differentiation. Dysfunction of epigenetic modifiers contributes significantly to the pathogenesis of AML. One frequently mutated gene involved in epigenetic modification is DNMT3A (DNA methyltransferase-3-alpha), a DNA methyltransferase that alters gene expression by de novo methylation of cytosine bases at CpG dinucleotides. Approximately 22% of AML and 36% of cytogenetically normal AML cases carry DNMT3A mutations and around 60% of these mutations affect the R882 codon. These mutations have been associated with poor prognosis and adverse survival outcomes for AML patients. Advances in whole-exome sequencing techniques have recently identified a large number of DNMT3A mutations present in clonal cells in normal elderly individuals with no features of haematological malignancy. Categorically distinct from other preleukaemic conditions, this disorder has been termed clonal haematopoiesis of indeterminate potential (CHIP). Further insight into the mutational landscape of CHIP may illustrate the consequence of particular mutations found in DNMT3A and identify specific "founder" mutations responsible for clonal expansion that may contribute to leukaemogenesis. This review will focus on current research and understanding of DNMT3A mutations in both AML and CHIP.

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

PubMed Central

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-01-01

Abstract Background Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

PubMed

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-03-01

Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Our results prove that d

An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

PubMed Central

Weitnauer, Gabriele; Gaisser, Sibylle; Trefzer, Axel; Stockert, Sigrid; Westrich, Lucy; Quiros, Luis M.; Mendez, Carmen; Salas, Jose A.; Bechthold, Andreas

2001-01-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA. PMID:11181344

An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tü57.

PubMed

Weitnauer, G; Gaisser, S; Trefzer, A; Stockert, S; Westrich, L; Quiros, L M; Mendez, C; Salas, J A; Bechthold, A

2001-03-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: Effect of sex and arsenic exposure

PubMed Central

Huang, Madelyn C.; Douillet, Christelle; Su, Mingming; Zhou, Kejun; Wu, Tao; Chen, Wenlian; Galanko, Joseph A.; Drobná, Zuzana; Saunders, R. Jesse; Martin, Elizabeth; Fry, Rebecca C.; Jia, Wei; Stýblo, Miroslav

2016-01-01

Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex-specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation. PMID:26883664

Modulation of microRNA expression in human lung cancer cells by the G9a histone methyltransferase inhibitor BIX01294

PubMed Central

PANG, ALAN LAP-YIN; TITLE, ALEXANDRA C.; RENNERT, OWEN M.

2014-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor-promoting or -suppressing effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly investigated. The present study examined the expression pattern of miRNAs in the non-small cell lung cancer cell line, H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono-and di-methylation of the lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, two miRNAs were identified that showed consistent downregulation following BIX01294 treatment. The results indicate that histone H3 methylation regulates miRNA expression in lung cancer cells, which may provide additional insight for future chemical treatment of lung cancer. PMID:24932239

Discovery of reversible DNA methyltransferase and lysine methyltransferase G9a inhibitors with antitumoral in vivo efficacy.

PubMed

Rabal, Obdulia; San José-Eneriz, Edurne; Agirre, Xabier; Sánchez-Arias, Juan A; Vilas-Zornoza, Amaia; Ugarte, Ana; De Miguel, Irene; Miranda, Estibaliz; Garate, Leire; Fraga, Mario; Santamarina, Pablo; Fernandez Perez, Raul; Ordoñez, Raquel; Sáez, Elena; Roa, Sergio; Garcia-Barchino, Maria Jose; Martinez-Climent, Jose Angel; Liu, Yingying; Wu, Wei; Xu, Musheng; Prosper, Felipe; Oyarzabal, Julen

2018-06-28

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear anti-proliferative efficacies (GI50 values in the nanomolar range). Based on epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept (ref 53). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human AML (Acute Myeloid Leukemia) xenograft in a mouse model.

Substrate Specificity of Human Protein Arginine Methyltransferase 7 (PRMT7)

PubMed Central

Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G.

2014-01-01

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-NG-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. PMID:25294873

Effect of Mild Acid on Gene Expression in Staphylococcus aureus

PubMed Central

Weinrick, Brian; Dunman, Paul M.; McAleese, Fionnuala; Murphy, Ellen; Projan, Steven J.; Fang, Yuan; Novick, Richard P.

2004-01-01

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, ∼5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus. PMID:15576791

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

PubMed Central

Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2015-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

Uncovering co-expression gene network modules regulating fruit acidity in diverse apples.

PubMed

Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Zhong, Gan-Yuan; Xu, Kenong

2015-08-16

Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5-81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a

Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a.

PubMed

An, Phan Nguyen Thuy; Shimaji, Kouhei; Tanaka, Ryo; Yoshida, Hideki; Kimura, Hiroshi; Fukusaki, Eiichiro; Yamaguchi, Masamitsu

2017-08-04

Epigenetics is now emerging as a key regulation in response to various stresses. We herein identified the Drosophila histone methyltransferase G9a (dG9a) as a key factor to acquire tolerance to starvation stress. The depletion of dG9a led to high sensitivity to starvation stress in adult flies, while its overexpression induced starvation stress resistance. The catalytic domain of dG9a was not required for starvation stress resistance. dG9a plays no apparent role in tolerance to other stresses including heat and oxidative stresses. Metabolomic approaches were applied to investigate global changes in the metabolome due to the loss of dG9a during starvation stress. The results obtained indicated that dG9a plays an important role in maintaining energy reservoirs including amino acid, trehalose, glycogen, and triacylglycerol levels during starvation. Further investigations on the underlying mechanisms showed that the depletion of dG9a repressed starvation-induced autophagy by controlling the expression level of Atg8a, a critical gene for the progression of autophagy, in a different manner to that in cancer cells. These results indicate a positive role for dG9a in starvation-induced autophagy.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

PubMed

Moore, M H; Gulbis, J M; Dodson, E J; Demple, B; Moody, P C

1994-04-01

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

PubMed

Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

2017-06-01

Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava.

PubMed

Wei, Yunxie; Liu, Guoyin; Bai, Yujing; Xia, Feiyu; He, Chaozu; Shi, Haitao; Foyer, Christine

2017-10-13

Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome

PubMed Central

Decewicz, Przemyslaw; Radlinska, Monika; Dziewit, Lukasz

2017-01-01

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity. PMID:28672885

Differential DNA methylation of the meiosis-specific gene FKBP6 in testes of yak and cattle-yak hybrids.

PubMed

Li, B; Luo, H; Weng, Q; Wang, S; Pan, Z; Xie, Z; Wu, W; Liu, H; Li, Q

2016-12-01

FK506-binding protein 6 (FKBP6) is essential for meiosis during mammalian spermatogenesis. However, the molecular regulation of FKBP6 during spermatogenesis remains unclear. In the present study, we performed molecular characterization of the meiosis-specific gene FKBP6 in yak testes. Yak FKBP6 encodes a polypeptide of 295 amino acid residues with an FK506-binding domain (FKBP_C) and three tetratricopeptide repeat domains. The methylation level of the FKBP6 promoter in testes was significantly higher in cattle-yak with male sterility than in yak, and the FKBP6 promoter was methylated in liver tissues in which FKBP6 is not expressed. FKBP6 promoter activity was significantly decreased after treatment with the M.SssI methyltransferase in vitro. Furthermore, the FKBP6 gene was remarkably activated in bovine mammary epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine. Taken together, our results demonstrate for the first time that the FKBP6 promoter is differentially methylated in testes; together with the functional promoter analysis, this suggests that methylation of this promoter may contribute to cattle-yak male infertility. © 2016 Blackwell Verlag GmbH.

Isolation of DNA methyltransferase from plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ehrlich, K.; Malbroue, C.

1987-05-01

DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from (/sup 3/H)AdoMet incorporated into acid precipitablemore » material per h at 30/sup 0/). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30/sup 0/.« less

Nicotinic acid modulates Legionella pneumophila gene expression and induces virulence traits.

PubMed

Edwards, Rachel L; Bryan, Andrew; Jules, Matthieu; Harada, Kaoru; Buchrieser, Carmen; Swanson, Michele S

2013-03-01

In response to environmental fluctuations or stresses, bacteria can activate transcriptional and phenotypic programs to coordinate an adaptive response. The intracellular pathogen Legionella pneumophila converts from a noninfectious replicative form to an infectious transmissive form when the bacterium encounters alterations in either amino acid concentrations or fatty acid biosynthesis. Here, we report that L. pneumophila differentiation is also triggered by nicotinic acid, a precursor of the central metabolite NAD(+). In particular, when replicative L. pneumophila are treated with 5 mM nicotinic acid, the bacteria induce numerous transmissive-phase phenotypes, including motility, cytotoxicity toward macrophages, sodium sensitivity, and lysosome avoidance. Transcriptional profile analysis determined that nicotinic acid induces the expression of a panel of genes characteristic of transmissive-phase L. pneumophila. Moreover, an additional 213 genes specific to nicotinic acid treatment were altered. Although nearly 25% of these genes lack an assigned function, the gene most highly induced by nicotinic acid treatment encodes a putative major facilitator superfamily transporter, Lpg0273. Indeed, lpg0273 protects L. pneumophila from toxic concentrations of nicotinic acid as judged by analyzing the growth of the corresponding mutant. The broad utility of the nicotinic acid pathway to couple central metabolism and cell fate is underscored by this small metabolite's modulation of gene expression by diverse microbes, including Candida glabrata, Bordetella pertussis, Escherichia coli, and L. pneumophila.

Epigenetics provides a new generation of oncogenes and tumour-suppressor genes

PubMed Central

Esteller, M

2006-01-01

Cancer is nowadays recognised as a genetic and epigenetic disease. Much effort has been devoted in the last 30 years to the elucidation of the ‘classical' oncogenes and tumour-suppressor genes involved in malignant cell transformation. However, since the acceptance that major disruption of DNA methylation, histone modification and chromatin compartments are a common hallmark of human cancer, epigenetics has come to the fore in cancer research. One piece is still missing from the story: are the epigenetic genes themselves driving forces on the road to tumorigenesis? We are in the early stages of finding the answer, and the data are beginning to appear: knockout mice defective in DNA methyltransferases, methyl-CpG-binding proteins and histone methyltransferases strongly affect the risk of cancer onset; somatic mutations, homozygous deletions and methylation-associated silencing of histone acetyltransferases, histone methyltransferases and chromatin remodelling factors are being found in human tumours; and the first cancer-prone families arising from germline mutations in epigenetic genes, such as hSNF5/INI1, have been described. Even more importantly, all these ‘new' oncogenes and tumour-suppressor genes provide novel molecular targets for designed therapies, and the first DNA-demethylating agents and inhibitors of histone deacetylases are reaching the bedside of patients with haematological malignancies. PMID:16404435

The O-methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit.

PubMed

Yauk, Yar-Khing; Chagné, David; Tomes, Sumathi; Matich, Adam J; Wang, Mindy Y; Chen, Xiuyin; Maddumage, Ratnasiri; Hunt, Martin B; Rowan, Daryl D; Atkinson, Ross G

2015-06-01

Phenylpropenes, such as eugenol and trans-anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non-producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co-located with seven candidate genes for phenylpropene O-methyltransferases (MdoOMT1-7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over-expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Protein arginine Methyltransferase 8 gene is expressed in pluripotent stem cells and its expression is modulated by the transcription factor Sox2.

PubMed

Solari, Claudia; Echegaray, Camila Vázquez; Luzzani, Carlos; Cosentino, María Soledad; Waisman, Ariel; Petrone, María Victoria; Francia, Marcos; Sassone, Alina; Canizo, Jésica; Sevlever, Gustavo; Barañao, Lino; Miriuka, Santiago; Guberman, Alejandra

2016-04-22

Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (Prmt). Although Prmt1 is essential in development, its paralogue Prmt8 has been poorly studied. This gene was reported to be expressed in nervous system and involved in neurogenesis. In this work, we found that Prmt8 is expressed in mouse embryonic stem cells (ESC) and in induced pluripotent stem cells, and modulated along differentiation to neural precursor cells. We found that Prmt8 promoter activity is induced by the pluripotency transcription factors Oct4, Sox2 and Nanog. Moreover, endogenous Prmt8 mRNA levels were reduced in ESC transfected with Sox2 shRNA vector. As a whole, our results indicate that Prmt8 is expressed in pluripotent stem cells and its transcription is modulated by pluripotency transcription factors. These findings suggest that besides its known function in nervous system, Prmt8 could play a role in pluripotent stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

PubMed

Arun, Muthukrishnan; Subramanyam, Kondeti; Theboral, Jeevaraj; Sivanandhan, Ganeshan; Rajesh, Manoharan; Kapil Dev, Gnanajothi; Jaganath, Balusamy; Manickavasagam, Markandan; Girija, Shanmugam; Ganapathi, Andy

2014-02-01

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

Monoamine Oxidase A (MAOA) and Catechol-O-Methyltransferase (COMT) Gene Polymorphisms Interact with Maternal Parenting in Association with Adolescent Reactive Aggression but not Proactive Aggression: Evidence of Differential Susceptibility.

PubMed

Zhang, Wenxin; Cao, Cong; Wang, Meiping; Ji, Linqin; Cao, Yanmiao

2016-04-01

To date, whether and how gene-environment (G × E) interactions operate differently across distinct subtypes of aggression remains untested. More recently, in contrast with the diathesis-stress hypothesis, an alternative hypothesis of differential susceptibility proposes that individuals could be differentially susceptible to environments depending on their genotypes in a "for better and for worse" manner. The current study examined interactions between monoamine oxidase A (MAOA) T941G and catechol-O-methyltransferase (COMT) Val158Met polymorphisms with maternal parenting on two types of aggression: reactive and proactive. Moreover, whether these potential G × E interactions would be consistent with the diathesis-stress versus the differential susceptibility hypothesis was tested. Within the sample of 1399 Chinese Han adolescents (47.2 % girls, M age = 12.32 years, SD = 0.50), MAOA and COMT genes both interacted with positive parenting in their associations with reactive but not proactive aggression. Adolescents with T alleles/TT homozygotes of MAOA gene or Met alleles of COMT gene exhibited more reactive aggression when exposed to low positive parenting, but less reactive aggression when exposed to high positive parenting. These findings provide the first evidence for distinct G × E interaction effects on reactive versus proactive aggression and lend further support for the differential susceptibility hypothesis.

Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.

2013-01-01

Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes wemore » successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.« less

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

PubMed

McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2016-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana.

PubMed

Hong, Sunghyun; Song, Hae-Ryong; Lutz, Kerry; Kerstetter, Randall A; Michael, Todd P; McClung, C Robertson

2010-12-07

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

PubMed Central

Hong, Sunghyun; Lutz, Kerry; Kerstetter, Randall A.; Michael, Todd P.; McClung, C. Robertson

2010-01-01

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general. PMID:21097700

DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate.

PubMed

Li, Wen; Yu, Min; Luo, Suhui; Liu, Huan; Gao, Yuxia; Wilson, John X; Huang, Guowei

2013-07-01

The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0-200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20-40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth. Copyright © 2013 Elsevier Inc. All rights reserved.

Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin.

PubMed

Witek, Marta A; Conn, Graeme L

2014-09-01

The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m(1)A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m(1)A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m(1)A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical enzyme is a functional 16S rRNA (m(1)A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA Methyltransferase Activity Assays: Advances and Challenges

PubMed Central

Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

2016-01-01

DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

PubMed

Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns

Analysis of Natural and Induced Variation in Tomato Glandular Trichome Flavonoids Identifies a Gene Not Present in the Reference Genome[W][OPEN

PubMed Central

Kim, Jeongwoon; Matsuba, Yuki; Ning, Jing; Schilmiller, Anthony L.; Hammar, Dagan; Jones, A. Daniel; Pichersky, Eran; Last, Robert L.

2014-01-01

Flavonoids are ubiquitous plant aromatic specialized metabolites found in a variety of cell types and organs. Methylated flavonoids are detected in secreting glandular trichomes of various Solanum species, including the cultivated tomato (Solanum lycopersicum). Inspection of the sequenced S. lycopersicum Heinz 1706 reference genome revealed a close homolog of Solanum habrochaites MOMT1 3′/5′ myricetin O-methyltransferase gene, but this gene (Solyc06g083450) is missing the first exon, raising the question of whether cultivated tomato has a distinct 3′ or 3′/5′ O-methyltransferase. A combination of mining genome and cDNA sequences from wild tomato species and S. lycopersicum cultivar M82 led to the identification of Sl-MOMT4 as a 3′ O-methyltransferase. In parallel, three independent ethyl methanesulfonate mutants in the S. lycopersicum cultivar M82 background were identified as having reduced amounts of di- and trimethylated myricetins and increased monomethylated myricetin. Consistent with the hypothesis that Sl-MOMT4 is a 3′ O-methyltransferase gene, all three myricetin methylation defective mutants were found to have defects in MOMT4 sequence, transcript accumulation, or 3′-O-methyltransferase enzyme activity. Surprisingly, no MOMT4 sequence is found in the Heinz 1706 reference genome sequence, and this cultivar accumulates 3-methyl myricetin and is deficient in 3′-methyl myricetins, demonstrating variation in this gene among cultivated tomato varieties. PMID:25128240

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

Cancer.gov

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

Methylated nucleosides in tRNA and tRNA methyltransferases

PubMed Central

Hori, Hiroyuki

2014-01-01

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

High expression of DNA methyltransferases in primary human medulloblastoma.

PubMed

Pócza, T; Krenács, T; Turányi, E; Csáthy, J; Jakab, Z; Hauser, P

2016-01-01

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Crystal structure of the avilamycin resistance-conferring methyltransferase AviRa from Streptomyces viridochromogenes.

PubMed

Mosbacher, Tanja G; Bechthold, Andreas; Schulz, Georg E

2003-05-23

The emergence of antibiotic-resistant bacterial strains is a widespread problem in contemporary medical practice and drug design. It is therefore important to elucidate the underlying mechanism in each case. The methyltransferase AviRa from Streptomyces viridochromogenes mediates resistance to the antibiotic avilamycin, which is closely related to evernimicin, an oligosaccharide antibiotic that has been used in medical studies. The structure of AviRa was determined by X-ray diffraction at 1.5A resolution. Phases were obtained from one selenomethionine residue introduced by site-directed mutagenesis. The chain-fold is similar to that of most methyltransferases, although AviRa contains two additional helices as a specific feature. A putative-binding site for the cofactor S-adenosyl-L-methionine was derived from homologous structures. It agrees with the conserved pattern of interacting amino acid residues. AviRa methylates a specific guanine base within the peptidyltransferase loop of the 23S ribosomal RNA. Guided by the target, the enzyme was docked to the cognate ribosomal surface, where it fit well into a deep cleft without contacting any ribosomal protein. The two additional alpha-helices of AviRa filled a depression in the surface. Since the transferred methyl group of the cofactor is in a pocket beneath the enzyme surface, the targeted guanine base has to flip out for methylation.

Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre

2007-04-01

Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{submore » 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.« less

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

PubMed

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judge, Russell A.; Zhu, Haizhong; Upadhyay, Anup K.

SETD8 is a histone H4–K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

Gene coexpression network analysis of fruit transcriptomes uncovers a possible mechanistically distinct class of sugar/acid ratio-associated genes in sweet orange.

PubMed

Qiao, Liang; Cao, Minghao; Zheng, Jian; Zhao, Yihong; Zheng, Zhi-Liang

2017-10-30

The ratio of sugars to organic acids, two of the major metabolites in fleshy fruits, has been considered the most important contributor to fruit sweetness. Although accumulation of sugars and acids have been extensively studied, whether plants evolve a mechanism to maintain, sense or respond to the fruit sugar/acid ratio remains a mystery. In a prior study, we used an integrated systems biology tool to identify a group of 39 acid-associated genes from the fruit transcriptomes in four sweet orange varieties (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). We reanalyzed the prior sweet orange fruit transcriptome data, leading to the identification of 72 genes highly correlated with the fruit sugar/acid ratio. The majority of these sugar/acid ratio-related genes are predicted to be involved in regulatory functions such as transport, signaling and transcription or encode enzymes involved in metabolism. Surprisingly, only three of these sugar/acid ratio-correlated genes are weakly correlated with sugar level and none of them overlaps with the acid-associated genes. Weighted Gene Coexpression Network Analysis (WGCNA) has revealed that these genes belong to four modules, Blue, Grey, Brown and Turquoise, with the former two modules being unique to the sugar/acid ratio control. Our results indicate that orange fruits contain a possible mechanistically distinct class of genes that may potentially be involved in maintaining fruit sugar/acid ratios and/or responding to the cellular sugar/acid ratio status. Therefore, our analysis of orange transcriptomes provides an intriguing insight into the potentially novel genetic or molecular mechanisms controlling the sugar/acid ratio in fruits.

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

PubMed

Horowitz, Scott; Dirk, Lynnette M A; Yesselman, Joseph D; Nimtz, Jennifer S; Adhikari, Upendra; Mehl, Ryan A; Scheiner, Steve; Houtz, Robert L; Al-Hashimi, Hashim M; Trievel, Raymond C

2013-10-16

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation.

PubMed

Małecki, Jędrzej; Jakobsson, Magnus E; Ho, Angela Y Y; Moen, Anders; Rustan, Arild C; Falnes, Pål Ø

2017-10-27

Lysine methylation is an important and much-studied posttranslational modification of nuclear and cytosolic proteins but is present also in mitochondria. However, the responsible mitochondrial lysine-specific methyltransferases (KMTs) remain largely elusive. Here, we investigated METTL12, a mitochondrial human S -adenosylmethionine (AdoMet)-dependent methyltransferase and found it to methylate a single protein in mitochondrial extracts, identified as citrate synthase (CS). Using several in vitro and in vivo approaches, we demonstrated that METTL12 methylates CS on Lys-395, which is localized in the CS active site. Interestingly, the METTL12-mediated methylation inhibited CS activity and was blocked by the CS substrate oxaloacetate. Moreover, METTL12 was strongly inhibited by the reaction product S -adenosylhomocysteine (AdoHcy). In summary, we have uncovered a novel human mitochondrial KMT that introduces a methyl modification into a metabolic enzyme and whose activity can be modulated by metabolic cues. Based on the established naming nomenclature for similar enzymes, we suggest that METTL12 be renamed CS-KMT (gene name CSKMT ). © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Physiological Study on Association between Nicotinamide N-Methyltransferase Gene Polymorphisms and Hyperlipidemia

PubMed Central

Zhu, Xiao-Juan; Lin, Ya-Jun; Chen, Wei; Wang, Ya-Hui; Qiu, Li-Qiang; Cai, Can-Xin; Xiong, Qun; Chen, Fei; Chen, Li-Hui; Zhou, Qiong

2016-01-01

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide. Our previous works indicate that NNMT is involved in the body mass index and energy metabolism, and recently the association between a SNP (rs694539) of NNMT and a variety of cardiovascular diseases was reported. At present, more than 200 NNMT single nucleotide polymorphisms (SNPs) have been identified in the databases of the human genome projects; however, the association between rs694539 variation and hyperlipidemia has not been reported yet, and whether there are any SNPs in NNMT significantly associated with hyperlipidemia is still unclear. In this paper, we selected 19 SNPs in NNMT as the tagSNPs using Haploview software (Haploview 4.2) first and then performed a case-control study to observe the association between these tagSNPs and hyperlipidemia and finally applied physiological approaches to explore the possible mechanisms through which the NNMT polymorphism induces hyperlipidemia. The results show that a SNP (rs1941404) in NNMT is significantly associated with hyperlipidemia, and the influence of rs1941404 variation on the resting energy expenditure may be the possible mechanism for rs1941404 variation to induce hyperlipidemia. PMID:27999813

Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiangbo; Zhang, Lu; Ding, Nianhua

2015-05-29

Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possessmore » efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.« less

Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

PubMed

Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

2018-06-01

Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

[Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

PubMed

Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

2014-09-01

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

PubMed Central

Gopal, J; Yebra, M J; Bhagwat, A S

1994-01-01

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework. Images PMID:7971279

Polysomnographic and neurometabolic features may mark preclinical autosomal dominant cerebellar ataxia, deafness, and narcolepsy due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1.

PubMed

Moghadam, Keivan Kaveh; Pizza, Fabio; Tonon, Caterina; Lodi, Raffaele; Carelli, Valerio; Poli, Francesca; Franceschini, Christian; Barboni, Piero; Seri, Marco; Ferrari, Simona; La Morgia, Chiara; Testa, Claudia; Cornelio, Ferdinando; Liguori, Rocco; Winkelmann, Juliane; Lin, Ling; Mignot, Emmanuel; Plazzi, Giuseppe

2014-05-01

We aimed to report the clinical picture of two asymptomatic daughters of a patient with autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN) due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1. Clinical assessment based on history, neurologic examination, sleep recordings, neurophysiologic neuroimaging, and genetic tests was performed. History and neurologic examination in both subjects were unremarkable. Genetic analysis disclosed in both the paternally-inherited heterozygous point mutation in the DNMT1 gene. Sleep recordings found sleep-onset rapid eye movement periods (SOREMPs) and proton magnetic resonance spectroscopy (MRS) revealed increased cerebellar myoinositol (mI) in both subjects. Auditory and ophthalmologic investigations as well as structural brain magnetic resonance imaging (MRI) scans revealed no abnormalities. The two asymptomatic carriers of the heterozygous DNMT1 mutation for ADCA-DN, a late-onset neurodegenerative disease, presented with SOREMPs associated with an increase of mI in the brain, a marker of glial cell activity and density characteristic of early stages of neurodegenerative diseases. Therefore, SOREMPs may precede the clinical picture of ADCA-DN as an early polysomnographic marker of central nervous system involvement detected by MRS. Copyright © 2014 Elsevier B.V. All rights reserved.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.

PubMed

Gonsalvez, Graydon B; Tian, Liping; Ospina, Jason K; Boisvert, François-Michel; Lamond, Angus I; Matera, A Gregory

2007-08-27

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.

Human nutrigenomics of gene regulation by dietary fatty acids.

PubMed

Afman, Lydia A; Müller, Michael

2012-01-01

Nutrigenomics employs high-throughput genomics technologies to unravel how nutrients modulate gene and protein expression and ultimately influence cellular and organism metabolism. The most often-applied genomics technique so far is transcriptomics, which allows quantifying genome-wide changes in gene expression of thousands of genes at the same time in one sample. The performance of gene expression quantification requires sufficient high-quality homogenous cellular material, therefore research in healthy volunteers is restricted to biopsies from easy accessible tissues such as subcutaneous adipose tissue, skeletal muscle and intestinal biopsies or even more easily accessible cells such as peripheral blood mononuclear cells from blood. There is now significant evidence that fatty acids, in particular unsaturated fatty acids, exert many of their effects through modulation of gene transcription by regulating the activity of numerous transcription factors, including nuclear receptors such as peroxisome proliferator activated receptors, liver X receptor and sterol regulatory binding proteins. This review evaluates the human nutrigenomics studies performed on dietary fat since the initiation of nutrigenomics research around 10 years ago. Although the number of studies is still limited, all studies clearly suggest that changes in dietary fatty acids intake and composition can have a significant impact on cellular adaptive response capacity by gene transcription changes in humans. This adds important knowledge to our understanding of the strong effects that various fatty acids can have on numerous metabolic and inflammatory pathways, signaling routes and homeostatic control in the cell and ultimately on whole body health. It is important to use and integrate nutrigenomics in all future nutrition studies to build up the necessary framework for evidence-based nutrition in near future. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

The Flexible Mind Is Associated with the Catechol-O-Methyltransferase (COMT) Val[superscript 158]Met Polymorphism: Evidence for a Role of Dopamine in the Control of Task-Switching

ERIC Educational Resources Information Center

Colzato, Lorenza S.; Waszak, Florian; Nieuwenhuis, Sander; Posthuma, Danielle; Hommel, Bernhard

2010-01-01

Genetic variability related to the catechol-O-methyltransferase (COMT) gene Val[superscript 128]Met polymorphism) has received increasing attention as a possible modulator of cognitive control functions. Recent evidence suggests that the Val[superscript 128]Met genotype may differentially affect cognitive stability and flexibility, in such a way…

Properly Substituted Analogues of BIX-01294 Lose Inhibition of G9a Histone Methyltransferase and Gain Selective Anti-DNA Methyltransferase 3A Activity

PubMed Central

Rotili, Dante; Tarantino, Domenico; Marrocco, Biagina; Gros, Christina; Masson, Véronique; Poughon, Valérie; Ausseil, Fréderic; Chang, Yanqi; Labella, Donatella; Cosconati, Sandro; Di Maro, Salvatore; Novellino, Ettore; Schnekenburger, Michael; Grandjenette, Cindy; Bouvy, Celine; Diederich, Marc; Cheng, Xiaodong; Arimondo, Paola B.; Mai, Antonello

2014-01-01

Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency. PMID:24810902

Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

PubMed

An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

2015-03-01

Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Site-specific bioalkylation of rapamycin by the RapM 16-O-methyltransferase.

PubMed

Law, Brian J C; Struck, Anna-Winona; Bennett, Matthew R; Wilkinson, Barrie; Micklefield, Jason

2015-05-01

The methylation of natural products by S -adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors in vitro and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple S -ethyl or S -allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches.

[Epigenetic heredity (deoxyribonucleic acid methylation): Clinical context in neurodegenerative disorders and ATXN2 gene].

PubMed

Laffita-Mesa, José Miguel; Bauer, Peter

2014-10-21

Epigenetics is the group of changes in the phenotype which are related with the process independently of the primary DNA sequence. These changes are intimately related with changes in the gene expression level and its profile across the body. These are mediated by histone tail modifications, DNA methylation, micro-RNAs, with chromatin remodeling remaining as the foundation of epigenetic changes. DNA methylation involves the covalent addition of methyl group to cytosine of the DNA, which is mediated by methyltransferases enzymes. DNA methylation regulates gene expression by repressing transcription, while de-methylation activates gene transcription. Several human diseases are related with the epigenetic process: cancer, Alzheimer disease, stroke, Parkinson disease, and diabetes. We present here the basis of epigenetic inheritance and show the pathogenic mechanisms relating epigenetics in human diseases, specifically with regard to neurodegeneration. We discuss current concepts aimed at understanding the contribution of epigenetics to human neurodegenerative diseases. We also discuss recent findings obtained in our and other centers regarding the ATXN2 gene that causes spinocerebellar ataxia 2 and amyotrophic lateral sclerosis. Epigenetics play a pivotal role in the pathogenesis of human diseases and in several neurodegenerative disorders, and this knowledge will illuminate the pathways in the diagnostic and therapeutic field, which ultimately will be translated into the clinic context of neurodegenerative diseases. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

PubMed

Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

2015-12-01

In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional and Structural Analysis of Phenazine O-Methyltransferase LaPhzM from Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin.

PubMed

Jiang, Jiasong; Guiza Beltran, Daisy; Schacht, Andrew; Wright, Stephen; Zhang, Limei; Du, Liangcheng

2018-04-20

Myxin is a well-known antibiotic that had been used for decades. It belongs to the phenazine natural products that exhibit various biological activities, which are often dictated by the decorating groups on the heteroaromatic three-ring system. The three rings of myxin carry a number of decorations, including an unusual aromatic N5, N10-dioxide. We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of myxin, and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative hydroxylation and aromatic N-oxidations of PDC to produce iodinin (1.6-dihydroxy- N5, N10-dioxide phenazine). In this work, we identified the LaPhzM gene from Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAM-dependent O-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is responsible for both monomethoxy and dimethoxy formation in all phenazine compounds isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing O-methylation of phenazines with non-, mono-, or di- N-oxide. In addition, we demonstrated a one-pot biosynthesis of myxin by in vitro reconstitution of the three phenazine-ring decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity and selectivity of the first characterized phenazine O-methyltransferase. These results will facilitate future exploitation of the thousands of phenazines as new antibiotics through metabolic engineering and chemoenzymatic syntheses.

Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence

PubMed Central

Shafrin, Farhana; Ferdous, Ahlan Sabah; Sarkar, Suprovath Kumar; Ahmed, Rajib; Amin, Al-; Hossain, Kawsar; Sarker, Mrinmoy; Rencoret, Jorge; Gutiérrez, Ana; del Rio, Jose C.; Sanan-Mishra, Neeti; Khan, Haseena

2017-01-01

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16–25% reduction in acid insoluble lignin for the whole stem and ~13–14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition. PMID:28051165

Purification and characterization of rice DNA methyltransferase.

PubMed

Teerawanichpan, Prapapan; Krittanai, Palika; Chauvatcharin, Nopmanee; Narangajavana, Jarunya

2009-08-01

Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160-170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development.

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of largemore » heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.« less

Association of polyunsaturated fatty acids in breast milk with fatty acid desaturase gene polymorphisms among Chinese lactating mothers.

PubMed

Ding, Zhen; Liu, Guo-Liang; Li, Xiang; Chen, Xue-Yan; Wu, Yi-Xia; Cui, Can-Can; Zhang, Xi; Yang, Guang; Xie, Lin

2016-06-01

The fatty acid desaturase (FADS) controls polyunsaturated fatty acid (PUFA) synthesis in human tissues and breast milk. Evaluate the influence of 10 single nucleotide polymorphisms (SNPs) and various haplotypes in the FADS gene cluster (FADS1, FADS2, FADS3) on PUFA concentration in the breast milk of 209 healthy Chinese women. PUFA concentrations were measured in breast milk using gas chromatography and genotyping was performed using the Sequenom Mass Array system. A SNP (rs1535) and 2-locus haplotypes (rs3834458-rs1535, rs1535-rs174575) in the FADS2 gene were associated with concentrations of γ-linoleic acid (GLA) and arachidonic acid (AA) in breast milk. Likewise, in the FADS1 gene, a 2-locus constructed haplotype (rs174547-rs174553) also affected GLA and AA concentration (P<0.05 for all). Minor allele carriers of the SNP and haplotypes described above had lower concentrations of GLA and AA. In the FADS2 gene, the 3-locus haplotype rs3834458-rs1535-rs174575, significantly affected concentrations of GLA but not AA. Pairwise comparison showed that individuals major homozygous for the SNP rs1000778 in the FADS3 gene had lower concentrations of ALA and linoleic acid (LA) in their breast milk. Polymorphisms in the FADS gene cluster influence PUFA concentrations in the breast milk of Chinese Han lactating women. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells.

PubMed

Steward, N; Kusano, T; Sano, H

2000-09-01

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

PubMed Central

Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

2007-01-01

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

Histone methyltransferase G9a modulates hepatic insulin signaling via regulating HMGA1.

PubMed

Xue, Weili; Huang, Jin; Chen, Hong; Zhang, Yu; Zhu, Xiuqin; Li, Jianshuang; Zhang, Wenquan; Yuan, Yangmian; Wang, Yan; Zheng, Ling; Huang, Kun

2018-02-01

Hepatic insulin sensitivity is critical for glucose homeostasis, and insulin resistance is a fundamental syndrome found in various metabolic disorders, including obesity and type 2 diabetes. Despite considerable studies on the mechanisms of hepatic insulin resistance, the link between epigenetic regulation and the development of insulin resistance remains elusive. Here, we reported that G9a/EHMT2, a histone methyltransferase, was markedly decreased in the liver of db/db mice and high-fat diet (HFD)-fed mice. In cultured hepatic cells, G9a knockdown resulted in downregulation of insulin receptor, p-AKT and p-GSK3β; while upon upregulation, G9a prevented the palmitic acid- or glucosamine-induced insulin resistance by preserving the normal level of insulin receptor and integrity of insulin signaling. Further mechanistic study suggested that G9a regulated the expression level of high mobility group AT-hook 1 (HMGA1), a key regulator responsible for the transcription of insulin receptor (INSR) gene. Overexpression of HMGA1 normalized the impaired insulin signaling in G9a knockdown hepatic cells. Importantly, in db/db mice, restoring the expression level of G9a not only upregulated HMGA1 level and improved the impaired hepatic insulin signaling, but also alleviated hyperglycemia and hyperinsulinemia. Together, our results revealed a novel role for G9a in modulating insulin signaling, at least in part, depending on its regulatory function on HMGA1. Copyright © 2017 Elsevier B.V. All rights reserved.

Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

Characterization of regioselective flavonoid O-methyltransferase from the Streptomyces sp. KCTC 0041BP.

PubMed

Darsandhari, Sumangala; Dhakal, Dipesh; Shrestha, Biplav; Parajuli, Prakash; Seo, Joo-Hyun; Kim, Tae-Su; Sohng, Jae Kyung

2018-06-01

A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-β-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4'-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C 2 and the C 3 and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented. Copyright © 2018 Elsevier Inc. All rights reserved.

Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

PubMed Central

Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

2013-01-01

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

N-3 polyunsaturated fatty acid regulation of hepatic gene transcription

PubMed Central

Jump, Donald B.

2009-01-01

Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

PubMed Central

Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

2015-01-01

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2.

PubMed

Eggert, Erik; Hillig, Roman C; Koehr, Silke; Stöckigt, Detlef; Weiske, Jörg; Barak, Naomi; Mowat, Jeffrey; Brumby, Thomas; Christ, Clara D; Ter Laak, Antonius; Lang, Tina; Fernandez-Montalvan, Amaury E; Badock, Volker; Weinmann, Hilmar; Hartung, Ingo V; Barsyte-Lovejoy, Dalia; Szewczyk, Magdalena; Kennedy, Steven; Li, Fengling; Vedadi, Masoud; Brown, Peter J; Santhakumar, Vijayaratnam; Arrowsmith, Cheryl H; Stellfeld, Timo; Stresemann, Carlo

2016-05-26

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.

Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

PubMed Central

Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

2014-01-01

Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression. PMID:25255382

Effects of oils rich in linoleic and α-linolenic acids on fatty acid profile and gene expression in goat meat.

PubMed

Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

2014-09-24

Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression.

Catechol-O-methyltransferase gene variants may associate with negative symptom response and plasma concentrations of prolactin in schizophrenia after amisulpride treatment.

PubMed

Chen, Chun-Yen; Yeh, Yi-Wei; Kuo, Shin-Chang; Ho, Pei-Shen; Liang, Chih-Sung; Yen, Che-Hung; Lu, Ru-Band; Huang, San-Yuan

2016-03-01

Catechol-O-methyltransferase (COMT) enzyme is involved in the pathogenesis of psychotic symptoms and may be associated with a therapeutic response to antipsychotic drugs. The aim of this study was to examine the relationship between COMT variants, plasma prolactin level, and the therapeutic effectiveness of amisulpride treatment in patients with schizophrenia. A 12-week naturalistic study of amisulpride treatment was carried out in 185 Han Chinese patients with schizophrenia. The patients were screened for 14 single-nucleotide polymorphisms of the COMT gene. The Positive and Negative Syndrome Scale (PANSS) was used to assess the improvement of psychopathological symptoms from the baseline to the end point in each subject. For better presentation of time-course changes in response status, a mixed model for repeated-measures (MMRM) analysis of symptom improvement during the 12-week treatment period was conducted. The change in plasma prolactin level after amisulpride treatment was also examined (n=51). No significant differences in the genotype frequencies of the COMT variants investigated were observed between responders and non-responders. Moreover, an MMRM analysis of psychopathological symptom improvement during the 12-week treatment course showed that it depended significantly on COMT variants (rs4680, rs4633, and rs6267), particularly regarding changes in negative symptoms. The increase in plasma prolactin levels observed was influenced by the COMT rs4680 variant and was positively correlated with a reduction in PANSS negative scores. Our results suggest that variation of the COMT gene is associated with treatment response regarding negative symptoms and prolactin changes after amisulpride treatment in patients with schizophrenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combining eicosapentaenoic acid, decosahexaenoic acid and arachidonic acid, using a fully crossed design, affect gene expression and eicosanoid secretion in salmon head kidney cells in vitro.

PubMed

Holen, Elisabeth; He, Juyun; Espe, Marit; Chen, Liqiou; Araujo, Pedro

2015-08-01

Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses. Copyright © 2015 Elsevier Ltd. All rights reserved.

SET DOMAIN GROUP 708, a histone H3 lysine 36-specific methyltransferase, controls flowering time in rice (Oryza sativa).

PubMed

Liu, Bing; Wei, Gang; Shi, Jinlei; Jin, Jing; Shen, Ting; Ni, Ting; Shen, Wen-Hui; Yu, Yu; Dong, Aiwu

2016-04-01

As a key epigenetic modification, the methylation of histone H3 lysine 36 (H3K36) modulates chromatin structure and is involved in diverse biological processes. To better understand the language of H3K36 methylation in rice (Oryza sativa), we chose potential histone methylation enzymes for functional exploration. In particular, we characterized rice SET DOMAIN GROUP 708 (SDG708) as an H3K36-specific methyltransferase possessing the ability to deposit up to three methyl groups on H3K36. Compared with the wild-type, SDG708-knockdown rice mutants displayed a late-flowering phenotype under both long-day and short-day conditions because of the down-regulation of the key flowering regulatory genes Heading date 3a (Hd3a), RICE FLOWERING LOCUS T1 (RFT1), and Early heading date 1 (Ehd1). Chromatin immunoprecipitation experiments indicated that H3K36me1, H3K36me2, and H3K36me3 levels were reduced at these loci in SDG708-deficient plants. More importantly, SDG708 was able to directly target and effect H3K36 methylation on specific flowering genes. In fact, knockdown of SDG708 led to misexpression of a set of functional genes and a genome-wide decrease in H3K36me1/2/3 levels during the early growth stages of rice. SDG708 is a methyltransferase that catalyses genome-wide deposition of all three methyl groups on H3K36 and is involved in many biological processes in addition to flowering promotion. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita.

PubMed

Nunes, Alexandro Silva; Campos, Vicente Paulo; Mascarello, Alessandra; Stumpf, Taisa Regina; Chiaradia-Delatorre, Louise Domenghini; Machado, Alan Rodrigues Teixeira; Santos Júnior, Helvécio Martins; Yunes, Rosendo Augusto; Nunes, Ricardo José; Oliveira, Denilson Ferreira

2013-12-01

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC50) of 171 μg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC50 of 260 μg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ~50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita. Copyright © 2013 Elsevier Inc. All rights reserved.

Posttranslational Protein Modifications in Type 1 Diabetes - Genetic Studies with PCMT1, the Repair Enzyme Protein Isoaspartate Methyltransferase (PIMT) Encoding Gene

PubMed Central

Wägner, Ana M.; Cloos, Paul; Bergholdt, Regine; Eising, Stefanie; Brorsson, Caroline; Stalhut, Martin; Christgau, Stephan; Nerup, Jørn; Pociot, Flemming

2008-01-01

BACKGROUND: Posttranslational protein modifications have been implicated in the development of autoimmunity. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) repairs modified proteins and is encoded by PCMT1, located in a region linked to type 1 diabetes (T1D), namely IDDM5. AIM: To evaluate the association between genetic variations in the PCMT1 gene and T1D. METHODS: Firstly, PCMT1 was sequenced in 26 patients with T1D (linked to IDDM5) and 10 control subjects. The variations found in PCMT1 were then tested (alone and interacting with a functional polymorphism in SUMO4 and with HLA) for association with T1D in 253 families (using transmission disequilibrium test). In a third step, the association of the functional variation in PCMT1 (rs4816) with T1D was analyzed in 778 T1D patients and 749 controls (using chi-square test). In vitro promoter activity was assessed by transfecting INS-1E cells with PCMT1 promoter constructs and a reporter gene, with or without cytokine stimulation. RESULTS: Four polymorphisms in complete linkage disequilibrium were identified in PCMT1 (5' to the gene (rs11155676), exon 5 (rs4816) and exon 8 (rs7818 and rs4552)). In the whole cohort of 253 families, the allele associated with increased PIMT enzyme activity (rs4816, allele A) was less frequently transmitted to the affected than to the non-affected offspring (46% vs. 53%, p = 0.099). This finding was even more evident in the subset of families where the proband had high-risk SUMO4 (p = 0.069) or low-risk HLA (p = 0.086). Surprisingly, in the case-control study with 778 cases and 749 controls, an inverse trend was found (40.36% of patients and 36.98% of controls had the allele, p = 0.055). PCMT1 promoter activity increased with cytokine stimulation, but no differences were detected between the constructs adjacent to rs11155676. CONCLUSION: PCMT1 was virtually associated with T1D in groups defined by other risk genes (SUMO4 and HLA). A general association in a not further

Monomethylioarsenicals are substratres for human arsenic (+3 oxidation state) methyltransferase

EPA Science Inventory

Monomethylthioarsenicals are substrates for human arsenic (+3 oxida1tion state) methyltransferase Methylated thioarsenicals are structural analogs of methylated oxyarsenic in which one or more oxygen atom bound t...

Thr105Ile (rs11558538) polymorphism in the histamine N-methyltransferase (HNMT) gene and risk for Parkinson disease

PubMed Central

Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; García-Martín, Elena; Agúndez, José A.G.

2016-01-01

Abstract Background/aims: Several neuropathological, biochemical, and pharmacological data suggested a possible role of histamine in the etiopathogenesis of Parkinson disease (PD). The single nucleotide polymorphism (SNP) rs11558538 in the histamine N-methyltransferase (HNMT) gene has been associated with the risk of developing PD by several studies but not by some others. We carried out a systematic review that included all the studies published on PD risk related to the rs11558538 SNP, and we conducted a meta-analysis following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Methods: We used several databases to perform the systematic review, the software Meta-DiSc 1.1.1 to perform the meta-analysis of the eligible studies, and the Q-statistic to test heterogeneity between studies. Results: The meta-analysis included 4 eligible case–control association studies for the HNMT rs11558538 SNP and the risk for PD (2108 patients, 2158 controls). The frequency of the minor allele positivity showed a statistically significant association with a decreased risk for PD, both in the total series and in Caucasians. Although homozygosity for the minor allele did not reach statistical significance, the test for trend indicates the occurrence of a gene–dose effect. Global diagnostic odds ratios (95% confidence intervals) for rs11558538T were 0.61 (0.46–0.81) for the total group, and 0.63 (0.45–0.88) for Caucasian patients. Conclusion: The present meta-analysis confirms published evidence suggesting that the HNMT rs11558538 minor allele is related to a reduced risk of developing PD. PMID:27399132

Genotype status of the dopamine-related catechol-O-methyltransferase (COMT) gene corresponds with desirability of “unhealthy” foods

PubMed Central

Wallace, Deanna L.; Aarts, Esther; Uquillas, Federico d’Oleire; Dang, Linh C.; Greer, Stephanie M.; Jagust, William J.; D’Esposito, Mark

2015-01-01

The role of dopamine is extensively documented in weight regulation and food intake in both animal models and humans. Yet the role of dopamine has not been well studied in individual differences for food desirability. Genotype status of the dopamine-related catechol-O-methyltransferase (COMT) gene has been shown to influence dopamine levels, with greater COMT enzymatic activity in val/val individuals corresponding to greater degradation of dopamine. Decreased dopamine has been associated with poorer cognitive control and diminished goal-directed behavior in various behavioral paradigms. Additionally, dopaminergic-rich regions such as the frontal cortex and dorsal striatum have been shown to be important for supporting food-related decision-making. However, the role of dopamine, as assessed by COMT genotype status, in food desirability has not been fully explored. Therefore, we utilized an individual’s COMT genotype status (n=61) and investigated food desirability based on self-rated “healthy” and “unhealthy” food perceptions. Here we found val/val individuals (n=19) have greater desirability for self-rated “unhealthy” food items, but not self-rated “healthy” food items, as compared to val/met (n=24) and met/met (n=18) individuals (p<0.005). Utilizing an objective health measure for the food items, we also found val/val and val/met individuals have greater desirability for objectively defined “unhealthy” food items, as compared to met/met individuals (p<0.01). This work further substantiates a role of dopamine in food-related behaviors and more specifically in relationship to food desirability for “unhealthy” food items. PMID:25963102

Highly Iterated Palindromic Sequences (HIPs) and Their Relationship to DNA Methyltransferases

PubMed Central

Elhai, Jeff

2015-01-01

The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences. PMID:25789551

Highly Iterated Palindromic Sequences (HIPs) and Their Relationship to DNA Methyltransferases.

PubMed

Elhai, Jeff

2015-03-17

The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences.

Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapetaniou, Evangelia G.; Kotsifaki, Dina; Providaki, Mary

2007-01-01

The DNA methyltransferase M.BseCI from B. stearothermophilus was crystallized as a complex with its cognate DNA. Crystals belong to space group P6 and diffract to 2.5 Å resolution at a synchrotron source. The DNA methyltransferase M.BseCI from Bacillus stearothermophilus (EC 2.1.1.72), a 579-amino-acid enzyme, methylates the N6 atom of the 3′ adenine in the sequence 5′-ATCGAT-3′. M.BseCI was crystallized in complex with its cognate DNA. The crystals were found to belong to the hexagonal space group P6, with unit-cell parameters a = b = 87.0, c = 156.1 Å, β = 120.0° and one molecule in the asymmetric unit. Twomore » complete data sets were collected at wavelengths of 1.1 and 2.0 Å to 2.5 and 2.8 Å resolution, respectively, using synchrotron radiation at 100 K.« less

Complexity and Entropy Analysis of DNMT1 Gene

USDA-ARS?s Scientific Manuscript database

Background: The application of complexity information on DNA sequence and protein in biological processes are well established in this study. Available sequences for DNMT1 gene, which is a maintenance methyltransferase is responsible for copying DNA methylation patterns to the daughter strands durin...

Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

PubMed Central

Preston, R. A.; Reinagel, P. S.; Jones, E. W.

1992-01-01

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph(-)) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph(-) screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph(-) mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity. PMID:1628805

RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bewg, William P.; Poovaiah, Charleson; Lan, Wu

Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all lignocellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. Furthermore, these genes, caffeoyl-CoA O-methyltransferasemore » ( CCoAOMT), ferulate 5-hydroxylase ( F5H) and caffeic acid O-methyltransferase ( COMT), impact lignin content and/or composition.« less

RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

DOE PAGES

Bewg, William P.; Poovaiah, Charleson; Lan, Wu; ...

2016-12-20

Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all lignocellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. Furthermore, these genes, caffeoyl-CoA O-methyltransferasemore » ( CCoAOMT), ferulate 5-hydroxylase ( F5H) and caffeic acid O-methyltransferase ( COMT), impact lignin content and/or composition.« less

Examination of Signatures of Recent Positive Selection on Genes Involved in Human Sialic Acid Biology.

PubMed

Moon, Jiyun M; Aronoff, David M; Capra, John A; Abbot, Patrick; Rokas, Antonis

2018-03-28

Sialic acids are nine carbon sugars ubiquitously found on the surfaces of vertebrate cells and are involved in various immune response-related processes. In humans, at least 58 genes spanning diverse functions, from biosynthesis and activation to recycling and degradation, are involved in sialic acid biology. Because of their role in immunity, sialic acid biology genes have been hypothesized to exhibit elevated rates of evolutionary change. Consistent with this hypothesis, several genes involved in sialic acid biology have experienced higher rates of non-synonymous substitutions in the human lineage than their counterparts in other great apes, perhaps in response to ancient pathogens that infected hominins millions of years ago (paleopathogens). To test whether sialic acid biology genes have also experienced more recent positive selection during the evolution of the modern human lineage, reflecting adaptation to contemporary cosmopolitan or geographically-restricted pathogens, we examined whether their protein-coding regions showed evidence of recent hard and soft selective sweeps. This examination involved the calculation of four measures that quantify changes in allele frequency spectra, extent of population differentiation, and haplotype homozygosity caused by recent hard and soft selective sweeps for 55 sialic acid biology genes using publicly available whole genome sequencing data from 1,668 humans from three ethnic groups. To disentangle evidence for selection from confounding demographic effects, we compared the observed patterns in sialic acid biology genes to simulated sequences of the same length under a model of neutral evolution that takes into account human demographic history. We found that the patterns of genetic variation of most sialic acid biology genes did not significantly deviate from neutral expectations and were not significantly different among genes belonging to different functional categories. Those few sialic acid biology genes that

Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

PubMed

Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

2018-02-01

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

PubMed Central

Züst, Roland; Li, Shi-Hua; Xie, Xuping; Velumani, Sumathy; Chng, Melissa; Toh, Ying-Xiu; Zou, Jing; Dong, Hongping; Shan, Chao; Pang, Jassia; Qin, Cheng-Feng; Newell, Evan W.; Shi, Pei-Yong

2018-01-01

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. PMID:29298302

Anhydrobiosis vs. aging: comparative genomics of protein repair L-isoaspartyl methyltransferases in the sleeping chironomid. .

NASA Astrophysics Data System (ADS)

Gusev, Oleg; Kikawada, Takahiro; Shagimardanova, Elena; Suetsugu, Yoshitaka; Ayupov, Rustam

Origin of anhydrobiosis in the larvae of the sleeping chironomid Polypedilum vanderplanki represents unique example of set of evolutionary events in a single species, resulted in acquiring new ability allowing survival in extremely changeable environment. Complex comparative analysis of the genome of P. vanderplanki resulted in discovery of a set of features, including existence of the set of unique clusters of genes contributing in desiccation resistance. Surprisingly, in several cases, the genes mainly contributing to the formation of the molecular shield in the larvae are sleeping chironomid-specific and have no homology with genes from other insects, including P. nubifer - a chironomid from the same genus. Protein L-isoaspartyl methyltransferase (PIMT) acts on proteins that have been non-enzymatically damaged due to age, and partially restores aspartic residues, extending life of the polypeptides. PIMT a highly conserved enzyme present in nearly all eukaryotes, and microorganisms mostly in a single copy (or in a few isoforms in certain plants and some bacteria). While conducting a comparative analysis of the genomes of two chironomid midge species different in their ability to stand complete water loss, we have noticed that structure and number of PIMT-coding genes in the desiccation resistant (anhydrobiotic) midge (Polypedilum vanderplanki, Pv) is different from those of the common desiccation-sensitive midge (Polypedilum nubifer, Pn) and the rest of insects. Both species have a clear orthologous PIMT shared by all insects. At the same time, in contrast to Pn which has only one PIMT gene (PnPimt-1), the Pv genome contains 12 additional genes paralogous to Pimt1 (PvPimt-2-12) presumably coding functional PIMT proteins, which are arranged in a single cluster. Remarkably, PvPimt-1 location in the Pv is different from the rest of Pimt-like genes. PvPimt-1 gene is ubiquitously expressed during the life cycle, but expression of the PvPimt2-12 is limited to the eggs

Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.

PubMed

Schmidt, Andreas; Marescau, Bart; Boehm, Ernest A; Renema, W Klaas Jan; Peco, Ruben; Das, Anib; Steinfeld, Robert; Chan, Sharon; Wallis, Julie; Davidoff, Michail; Ullrich, Kurt; Waldschütz, Ralph; Heerschap, Arend; De Deyn, Peter P; Neubauer, Stefan; Isbrandt, Dirk

2004-05-01

We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.

Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

PubMed

Zhu, Feng; Xi, De-Hui; Yuan, Shu; Xu, Fei; Zhang, Da-Wei; Lin, Hong-Hui

2014-06-01

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

PubMed Central

Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

2015-01-01

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid.

PubMed

Mira, Nuno P; Palma, Margarida; Guerreiro, Joana F; Sá-Correia, Isabel

2010-10-25

Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic

How to consistently link extraversion and intelligence to the catechol-O-methyltransferase (COMT) gene: on defining and measuring psychological phenotypes in neurogenetic research.

PubMed

Wacker, Jan; Mueller, Erik M; Hennig, Jürgen; Stemmler, Gerhard

2012-02-01

The evidence for associations between genetic polymorphisms and complex behavioral/psychological phenotypes (traits) has thus far been weak and inconsistent. Using the well-studied Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene as an example, we demonstrate that using theoretical models to guide phenotype definition and measuring the phenotypes of interest with a high degree of specificity reveals strong gene-behavior associations that are consistent with prior work and that would have otherwise gone unnoticed. Only after statistically controlling for irrelevant portions of phenotype variance did we observe strong (Cohen's d = 0.33-0.70) and significant associations between COMT Val158Met and both cognitive and affective traits in a healthy male sample (N = 201) in Study 1: Carriers of the Met allele scored higher in fluid intelligence (reasoning) but lower in both crystallized intelligence (general knowledge) and the agency facet of extraversion. In Study 2, we conceptually replicated the association of COMT Val158Met with the agency facet of extraversion after partialing irrelevant phenotype variance in a female sample (N = 565). Finally, through reanalysis of a large published data set we showed that Met allele carriers also scored higher in indicators of fluid intelligence after partialing verbal fluency. Because the Met allele codes for a less efficient variant of the enzyme COMT, resulting in higher levels of extrasynaptic prefrontal dopamine, these observations provide further support for a role for dopamine in both intelligence and extraversion. More importantly, the present findings have important implications for the definition of psychological phenotypes in neurogenetic research.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India

PubMed Central

Wangkheimayum, Jayalaxmi; Paul, Deepjyoti; Dhar, Debadatta; Nepram, Rajlakshmi; Chetri, Shiela; Bhowmik, Deepshikha; Chakravarty, Atanu

2017-01-01

ABSTRACT The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum β-lactamases and metallo-β-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings. PMID:28320725

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis

PubMed Central

Jin, Junfei; Lu, Zhongyang; Li, Yanchun; Cowart, L. Ashley; Lopes-Virella, Maria F.

2018-01-01

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages. PMID:29474492

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis.

PubMed

Jin, Junfei; Lu, Zhongyang; Li, Yanchun; Cowart, L Ashley; Lopes-Virella, Maria F; Huang, Yan

2018-01-01

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages.

A Type III Protein Arginine Methyltransferase from the Protozoan Parasite Trypanosoma brucei*

PubMed Central

Fisk, John C.; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G.; Read, Laurie K.

2009-01-01

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle. PMID:19254949

A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.

PubMed

Fisk, John C; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G; Read, Laurie K

2009-04-24

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

PubMed

Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

2000-12-07

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Donglin; Qu, Xiying; Li, Lin

Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was foundmore » to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency.« less

Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

PubMed

Wiley, Kenneth L; Treadwell, Edward; Manigaba, Kayihura; Word, Beverly; Lyn-Cook, Beverly D

2013-02-01

Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in

DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA).

PubMed

Erova, Tatiana E; Kosykh, Valeri G; Sha, Jian; Chopra, Ashok K

2012-05-01

Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam(+)) and GM33 (∆dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA(+) strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ∆gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation

Biotechnological Production of Dimethoxyflavonoids Using a Fusion Flavonoid O-Methyltransferase Possessing Both 3'- and 7-O-Methyltransferase Activities.

PubMed

Lee, Danbi; Park, Hye Lin; Lee, Sang-Won; Bhoo, Seong Hee; Cho, Man-Ho

2017-05-26

Although they are less abundant in nature, methoxyflavonoids have distinct physicochemical and pharmacological properties compared to common nonmethylated flavonoids. Thus, enzymatic conversion and biotransformation using genetically engineered microorganisms of flavonoids have been attempted for the efficient production of methoxyflavonoids. Because of their regiospecificity, more than two flavonoid O-methyltransferases (FOMTs) and enzyme reactions are required to biosynthesize di(or poly)-methoxyflavonoids. For the one-step biotechnological production of bioactive di-O-methylflavonoids, we generated a multifunctional FOMT fusing a 3'-OMT (SlOMT3) and a 7-OMT (OsNOMT). The SlOMT3/OsNOMT fusion enzyme possessed both 3'- and 7-OMT activities to diverse flavonoid substrates, which were comparable to those of individual SlOMT3 and OsNOMT. The SlOMT3/OsNOMT enzyme also showed 3'- and 7-OMT activity for 7- or 3'-O-methylflavonoids, respectively, suggesting that the fusion enzyme can sequentially methylate flavonoids into di-O-methylflavonoids. The biotransformation of the flavonoids quercetin, luteolin, eriodictyol, and taxifolin using SlOMT3/OsNOMT-transformed Escherichia coli generated corresponding di-O-methylflavonoids, rhamnazin, velutin, 3',7-di-O-methyleriodictyol, and 3',7-di-O-methyltaxifolin, respectively. These results indicate that dimethoxyflavonoids may be efficiently produced from nonmethylated flavonoid precursors through a one-step biotransformation using the engineered E. coli harboring the SlOMT3/OsNOMT fusion gene.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

PubMed

Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

2016-06-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. © 2016 American Society of Plant Biologists. All Rights Reserved.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm ofmore » the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.« less

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase.

PubMed

Debler, Erik W; Jain, Kanishk; Warmack, Rebeccah A; Feng, You; Clarke, Steven G; Blobel, Günter; Stavropoulos, Pete

2016-02-23

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

PubMed Central

Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

2016-01-01

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs. PMID:26858449

Egg-specific expression of protein with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis.

PubMed

Kim, Seon-Hee; Cho, Hye-Jeong; Sohn, Woon-Mok; Ahn, Chun-Seob; Kong, Yoon; Yang, Hyun-Jong; Bae, Young-An

2015-08-01

Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase 2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both of which were elevated in the C. sinensis eggs. However, CsDNMT2 and 5 mC were marginally detected in other histological regions of C. sinensis adults including ovaries and seminal receptacle. The methylation site seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon. Taken together, our data strongly suggest that C. sinensis has preserved the functional DNA methylation machinery and that DNMT2 acts as a genuine alternative to DNMT1/DNMT3 to methylate DNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

EPA Science Inventory

MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption

PubMed Central

Montagnani, Marco; Abrahamsson, Anna; Gälman, Cecilia; Eggertsen, Gösta; Marschall, Hanns-Ulrich; Ravaioli, Elisa; Einarsson, Curt; Dawson, Paul A

2006-01-01

The etiology of most cases of idiopathic bile acid malabsorption (IBAM) is unknown. In this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR) and peroxisome proliferator activated receptor alpha (PPARα). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using 75Se-homocholic acid taurine (SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4). The ASBT, FXR, and PPARα genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC, and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARα genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM. PMID:17171805

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation.

PubMed

Ariyawutthiphan, Orapin; Ose, Toyoyuki; Minami, Atsushi; Shinde, Sandip; Sinde, Sandip; Tsuda, Muneya; Gao, Yong-Gui; Yao, Min; Oikawa, Hideaki; Tanaka, Isao

2012-11-01

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping1

PubMed Central

Lapierre, Catherine; Pollet, Brigitte; Petit-Conil, Michel; Toval, Gabriel; Romero, Javier; Pilate, Gilles; Leplé, Jean-Charles; Boerjan, Wout; Ferret, Valérie; De Nadai, Véronique; Jouanin, Lise

1999-01-01

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry. PMID:9880356

Docosahexaenoic Acid (DHA) and Hepatic Gene Transcription1,3

PubMed Central

Jump, Donald B.; Botolin, Daniela; Wang, Yun; Xu, Jinghua; Demeure, Olivier; Christian, Barbara

2008-01-01

The type and quantity of dietary fat ingested contributes to the onset and progression of chronic diseases, like diabetes and atherosclerosis. The liver plays a central role in whole body lipid metabolism and responds rapidly to changes in dietary fat composition. Polyunsaturated fatty acids (PUFA) play a key role in membrane composition and function, metabolism and the control of gene expression. Certain PUFA, like the n-3 PUFA, enhance hepatic fatty acid oxidation and inhibit fatty acid synthesis and VLDL secretion, in part, by regulating gene expression. Our studies have established that key transcription factors, like PPARα, SREBP-1, ChREBP and MLX, are regulated by n-3 PUFA, which in turn control levels of proteins involved in lipid and carbohydrate metabolism. Of the n-3 PUFA, 22:6,n-3 has recently been established as a key controller of hepatic lipid synthesis. 22:6,n-3 controls the 26S proteasomal degradation of the nuclear form of SREBP-1. SREBP-1 is a major transcription factor that controls the expression of multiple genes involved fatty acid synthesis and desaturation. 22:6,n-3 suppresses nuclear SREBP-1 which, in turn suppresses lipogenesis. This mechanism is achieved, in part, through control of the phosphorylation status of protein kinases. This review will examine both the general features of PUFA-regulated hepatic gene transcription and highlight the unique mechanisms by which 22:6,n-3 impacts gene expression. The outcome of this analysis will reveal that changes in hepatic 22:6,n-3 content has a major impact on hepatic lipid and carbohydrate metabolism. Moreover, the mechanisms involve 22:6,n-3 control of several well-known signaling pathways, such as Akt, Erk1/2, Gsk3β and PKC (novel or atypical). 22:6,n-3 control of these same signaling pathways in non-hepatic tissues may help explain the diverse actions of n-3 PUFA on such complex physiological processes as visual acuity and learning. PMID:18343222

DOT1L histone methyltransferase regulates the expression of BCAT1 and is involved in sphere formation and cell migration of breast cancer cell lines.

PubMed

Oktyabri, Dulamsuren; Ishimura, Akihiko; Tange, Shoichiro; Terashima, Minoru; Suzuki, Takeshi

2016-04-01

DOT1L is a histone H3 lysine 79 (H3K79) methyltransferase mainly implicated in leukemia. Here we analyzed the function of DOT1L in breast cancer cells. The expression of DOT1L was up-regulated in malignant breast cancer tissues. Over-expression of DOT1L significantly increased the sphere formation and the cell migration activities of MCF7 breast cancer cell line. In contrast, knockdown of DOT1L reduced the cell migration activity of MDA-MB-231 breast cancer cell line. BCAT1, which encodes a branched-chain amino acid transaminase, was identified as one of the target genes controlled by DOT1L through the regulation of H3K79 methylation. Mechanistic investigation revealed that BCAT1 might be an important regulator responsible for DOT1L-mediated sphere formation and cell migration in breast cancer cells. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity.

PubMed

Murphy, James; Klumpp, Jochen; Mahony, Jennifer; O'Connell-Motherway, Mary; Nauta, Arjen; van Sinderen, Douwe

2014-10-01

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return. Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages. SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culman, J.; Torda, T.; Weise, V.K.

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments ofmore » other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.« less

Inheritance of Adrenal Phenylethanolamine N-Methyltransferase Activity in the Rat

PubMed Central

Stolk, Jon M.; Vantini, Guido; Guchhait, Ras B.; Hurst, Jeffrey H.; Perry, Bruce D.; U'Prichard, David C.; Elston, Robert C.

1984-01-01

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes the S-adenosyl-l-methionine-dependent methylation of (-)norepinephrine to (-)epinephrine in the adrenal medulla. Adrenal PNMT activity is markedly different in two highly inbred rat strains; enzyme activity in the F344 strain is more than fivefold greater than that in the Buf strain. Initial characterization of the enzyme in the two inbred strains reveals evidence for catalytic and structural differences, as reflected in dissimilar Km values for the cosubstrate (S-adenosyl-l-methionine) and prominent differences in thermal inactivation curves. To assess adrenal PNMT activity in an F344 x Buf pedigree, we employed a statistical procedure to test for one- and two-locus hypotheses in the presence of within-class correlations due to cage or litter effects. The PNMT data in the pedigree are best accounted for by segregation at a simple major locus superimposed upon a polygenic background; data obtained from the biochemical studies suggest that the major locus is a structural gene locus. PMID:6149973

Dynamic changes in methylome and transcriptome patterns in response to methyltransferase inhibitor 5-azacytidine treatment in citrus

PubMed Central

Xu, Jidi; Wang, Xia; Cao, Hongbo; Xu, Haidan; Xu, Qiang

2017-01-01

Abstract DNA methylation is known to play an important role in various developmental processes in plants. However, there is a general lack of understanding about the possible functions of DNA methylation in fruit trees. Using callus as a model, methylome, transcriptome and metabolite changes were assessed after treatment with the DNA methyltransferase inhibitor 5-azacytidine (5azaC). Genome-wide methylome analysis revealed the demethylation of a diverse of genes, including many genes encoding transcription factors (TFs), genes involved in biological processes, and the up-regulation of a wide range of transposable elements (TEs). Combined with the RNA-seq data, we observed no obvious genome-wide correlation between the changes in methylation status and expression levels. Furthermore, 5azaC treatment induced carotenoid degradation along with strong activation of carotenoid cleavage dioxygenases 1 (CpCCD1). Functional complementation analysis in bacterial system showed that CpCCD1 exhibited strong catalytic activities toward zeaxanthin, β-carotene and lycopene. In summary, 5azaC treatments induced carotenoid degradation by CpCCD1 activation and led to a genome-wide demethylation effect. PMID:28575160

Enhanced detoxification and degradation of herbicide atrazine by a group of O-methyltransferases in rice.

PubMed

Lu, Yi Chen; Luo, Fang; Pu, Zhong Ji; Zhang, Shuang; Huang, Meng Tian; Yang, Hong

2016-12-01

Atrazine (ATR) as a toxic herbicide has become one of the seriously environmental contaminants worldwide due to its long-term intensive use in crop production. This study identified novel methyltransferases (MTs) involved in detoxification and degradation of ATR residues in rice plants. From a subset of MTs differentially expressed in ATR-exposed rice, forty-four O-methyltransferase genes were investigated. Total activities were significantly enhanced by ATR in rice tissues. To prove detoxifying capacity of the MTs in rice plants, two rice O-MTs (LOC_Os04g09604 and LOC_Os11g15040) were selected and transformed into yeast cells (Pichia pastoris X-33). The positive transformants accumulated less ATR and showed less toxicity. Using UPLC-TOF-MS/MS, ATR-degraded products in rice and yeast cells were characterized. A novel O-methylated-modified metabolite (atraton) and six other ATR-derivatives were detected. The topological interaction between LOC_Os04g09604 enzyme and its substrate was specially analyzed by homology modeling programs, which was well confirmed by the molecular docking analysis. The significance of the study is to provide a better understanding of mechanisms for the specific detoxification and degradation of ATR residues in rice growing in environmentally relevant ATR-contaminated soils and may hold a potential engineering perspective for generating ATR-resistant rice that helps to minimize ATR residues in crops. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biocomputional construction of a gene network under acid stress in Synechocystis sp. PCC 6803.

PubMed

Li, Yi; Rao, Nini; Yang, Feng; Zhang, Ying; Yang, Yang; Liu, Han-ming; Guo, Fengbiao; Huang, Jian

2014-01-01

Acid stress is one of the most serious threats that cyanobacteria have to face, and it has an impact at all levels from genome to phenotype. However, very little is known about the detailed response mechanism to acid stress in this species. We present here a general analysis of the gene regulatory network of Synechocystis sp. PCC 6803 in response to acid stress using comparative genome analysis and biocomputational prediction. In this study, we collected 85 genes and used them as an initial template to predict new genes through co-regulation, protein-protein interactions and the phylogenetic profile, and 179 new genes were obtained to form a complete template. In addition, we found that 11 enriched pathways such as glycolysis are closely related to the acid stress response. Finally, we constructed a regulatory network for the intricate relationship of these genes and summarize the key steps in response to acid stress. This is the first time a bioinformatic approach has been taken systematically to gene interactions in cyanobacteria and the elaboration of their cell metabolism and regulatory pathways under acid stress, which is more efficient than a traditional experimental study. The results also provide theoretical support for similar research into environmental stresses in cyanobacteria and possible industrial applications. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

USDA-ARS?s Scientific Manuscript database

Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

Dynamic regulation of six histone H3 lysine (K) methyltransferases in response to prolonged anoxia exposure in a freshwater turtle.

PubMed

Wijenayake, Sanoji; Hawkins, Liam J; Storey, Kenneth B

2018-04-05

The importance of histone lysine methylation is well established in health, disease, early development, aging, and cancer. However, the potential role of histone H3 methylation in regulating gene expression in response to extended periods of oxygen deprivation (anoxia) in a natural, anoxia-tolerant model system is underexplored. Red-eared sliders (Trachemys scripta elegans) can tolerate and survive three months of absolute anoxia and recover without incurring detrimental cellular damage, mainly by reducing the overall metabolic rate by 90% when compared to normoxia. Stringent regulation of gene expression is a vital aspect of metabolic rate depression in red-eared sliders, and as such we examined the anoxia-responsive regulation of histone lysine methylation in the liver during 5 h and 20 h anoxia exposure. Interestingly, this is the first study to illustrate the existence of histone lysine methyltransferases (HKMTs) and corresponding histone H3 lysine methylation levels in the liver of anoxia-tolerant red-eared sliders. In brief, H3K4me1, a histone mark associated with active transcription, and two corresponding histone lysine methyltransferases that modify H3K4me1 site, significantly increased in response to anoxia. On the contrary, H3K27me1, another transcriptionally active histone mark, significantly decreased during 20 h anoxia, and a transcriptionally repressive histone mark, H3K9me3, and the corresponding KMTs, similarly increased during 20 h anoxia. Overall, the results suggest a dynamic regulation of histone H3 lysine methylation in the liver of red-eared sliders that could theoretically aid in the selective upregulation of genes that are necessary for anoxia survival, while globally suppressing others to conserve energy. Copyright © 2018 Elsevier B.V. All rights reserved.

Biosynthesis and Heterologous Production of Vioprolides: Rational Biosynthetic Engineering and Unprecedented 4-Methylazetidinecarboxylic Acid Formation.

PubMed

Yan, Fu; Auerbach, David; Chai, Yi; Keller, Lena; Tu, Qiang; Hüttel, Stephan; Glemser, Amelie; Grab, Hanusch A; Bach, Thorsten; Zhang, Youming; Müller, Rolf

2018-04-25

Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35. So far, nothing is known about their biosynthesis, including the origin of the unusual 4 methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at single digit mg/L scale, we developed a stable host that eventually allowed for yields up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S-adenosylmethionine (SAM) dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, non-natural vioprolide derivatives were generated via rational genetic engineering. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

PubMed Central

Hassler, Melanie R.; Klisaroska, Aleksandra; Kollmann, Karoline; Steiner, Irene; Bilban, Martin; Schiefer, Ana-Iris; Sexl, Veronika; Egger, Gerda

2012-01-01

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use. PMID:22687603

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-11-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed Central

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-01-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla. Images PMID:2903502

Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

PubMed Central

Ren, Dacheng; Zuo, Rongjun; González Barrios, Andrés F.; Bedzyk, Laura A.; Eldridge, Gary R.; Pasmore, Mark E.; Wood, Thomas K.

2005-01-01

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 μg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 μg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 μg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 μg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid). PMID:16000817

A Panoptic Uncovering of the Dynamical Evolution of the Zika Virus NS5 Methyltransferase Binding Site Loops- Zeroing in on the Molecular Landscape.

PubMed

Devnarain, Nikita; Soliman, Mahmoud E S

2018-06-20

The global threat of the Zika virus to humanity is real. Innovative and potent anti-Zika virus drugs are still at large, due to the lack of anti-Zika virus drugs that have passed phase 1 trials. Experimental research has revealed novel inhibitors of Zika virus NS5 methyltransferase enzyme. This study has taken a step further to provide insight into the molecular dynamics of Zika virus and inhibitor binding, which have not been established experimentally. Movements of the methyltransferase binding site loops have a large role to play in the methylation of the viral mRNA cap, which is essential for Zika virus replication. Here we pinpoint the binding interactions between each potential inhibitor and the methyltransferase, residues that are responsible for binding, as well as which inhibitor-bound complex renders the methyltransferase more stable. We also highlight the conformational changes that occur within the methyltransferase to accommodate binding of inhibitors and consequences of those changes upon the RNA- and cap-binding sites in the methyltransferase. This research will improve the understanding of the Zika virus NS5 methyltransferase enzyme, and will be beneficial in driving the development of anti-Zika virus drugs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism

NASA Astrophysics Data System (ADS)

Ke, Zhang-Hong; Pan, Jie-Xue; Jin, Lu-Yang; Xu, Hai-Yan; Yu, Tian-Tian; Ullah, Kamran; Rahman, Tanzil Ur; Ren, Jun; Cheng, Yi; Dong, Xin-Yan; Sheng, Jian-Zhong; Huang, He-Feng

2016-08-01

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns.

Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism

PubMed Central

Ke, Zhang-Hong; Pan, Jie-Xue; Jin, Lu-Yang; Xu, Hai-Yan; Yu, Tian-Tian; Ullah, Kamran; Rahman, Tanzil Ur; Ren, Jun; Cheng, Yi; Dong, Xin-Yan; Sheng, Jian-Zhong; Huang, He-Feng

2016-01-01

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns. PMID:27502578

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

PubMed

Swinehart, William E; Henderson, Jeremy C; Jackman, Jane E

2013-08-01

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

The methyltransferase SMYD3 mediates the recruitment of transcriptional cofactors at the myostatin and c-Met genes and regulates skeletal muscle atrophy

PubMed Central

Proserpio, Valentina; Fittipaldi, Raffaella; Ryall, James G.; Sartorelli, Vittorio; Caretti, Giuseppina

2013-01-01

Elucidating the epigenetic mechanisms underlying muscle mass determination and skeletal muscle wasting holds the potential of identifying molecular pathways that constitute possible drug targets. Here, we report that the methyltransferase SMYD3 modulates myostatin and c-Met transcription in primary skeletal muscle cells and C2C12 myogenic cells. SMYD3 targets the myostatin and c-Met genes and participates in the recruitment of the bromodomain protein BRD4 to their regulatory regions through protein–protein interaction. By recruiting BRD4, SMYD3 favors chromatin engagement of the pause–release factor p-TEFb (positive transcription elongation factor) and elongation of Ser2-phosphorylated RNA polymerase II (PolIISer2P). Reducing SMYD3 decreases myostatin and c-Met transcription, thus protecting from glucocorticoid-induced myotube atrophy. Supporting functional relevance of the SMYD3/BRD4 interaction, BRD4 pharmacological blockade by the small molecule JQ1 prevents dexamethasone-induced myostatin and atrogene up-regulation and spares myotube atrophy. Importantly, in a mouse model of dexamethasone-induced skeletal muscle atrophy, SMYD3 depletion prevents muscle loss and fiber size decrease. These findings reveal a mechanistic link between SMYD3/BRD4-dependent transcriptional regulation, muscle mass determination, and skeletal muscle atrophy and further encourage testing of small molecules targeting specific epigenetic regulators in animal models of muscle wasting. PMID:23752591

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.

PubMed

Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

2011-02-04

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

DOE PAGES

Hao, Yue; Blair, Patricia M.; Sharma, Abhishek; ...

2015-01-30

Peptide antibiotics represent a class of conformationally-constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here in this paper, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide amore » rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).« less

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Yue; Blair, Patricia M.; Sharma, Abhishek

Peptide antibiotics represent a class of conformationally-constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here in this paper, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide amore » rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).« less

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Wenbin; Cui Zhihong; Ao Lin

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. Themore » prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.« less

Identifying new susceptibility genes on dopaminergic and serotonergic pathways for the framing effect in decision-making.

PubMed

Gao, Xiaoxue; Liu, Jinting; Gong, Pingyuan; Wang, Junhui; Fang, Wan; Yan, Hongming; Zhu, Lusha; Zhou, Xiaolin

2017-09-01

The framing effect refers the tendency to be risk-averse when options are presented positively but be risk-seeking when the same options are presented negatively during decision-making. This effect has been found to be modulated by the serotonin transporter gene (SLC6A4) and the catechol-o-methyltransferase gene (COMT) polymorphisms, which are on the dopaminergic and serotonergic pathways and which are associated with affective processing. The current study aimed to identify new genetic variations of genes on dopaminergic and serotonergic pathways that may contribute to individual differences in the susceptibility to framing. Using genome-wide association data and the gene-based principal components regression method, we examined genetic variations of 26 genes on the pathways in 1317 Chinese Han participants. Consistent with previous studies, we found that the genetic variations of the SLC6A4 gene and the COMT gene were associated with the framing effect. More importantly, we demonstrated that the genetic variations of the aromatic-L-amino-acid decarboxylase (DDC) gene, which is involved in the synthesis of both dopamine and serotonin, contributed to individual differences in the susceptibility to framing. Our findings shed light on the understanding of the genetic basis of affective decision-making. © The Author (2017). Published by Oxford University Press.

Identifying new susceptibility genes on dopaminergic and serotonergic pathways for the framing effect in decision-making

PubMed Central

Gao, Xiaoxue; Liu, Jinting; Gong, Pingyuan; Wang, Junhui; Fang, Wan; Yan, Hongming; Zhu, Lusha

2017-01-01

Abstract The framing effect refers the tendency to be risk-averse when options are presented positively but be risk-seeking when the same options are presented negatively during decision-making. This effect has been found to be modulated by the serotonin transporter gene (SLC6A4) and the catechol-o-methyltransferase gene (COMT) polymorphisms, which are on the dopaminergic and serotonergic pathways and which are associated with affective processing. The current study aimed to identify new genetic variations of genes on dopaminergic and serotonergic pathways that may contribute to individual differences in the susceptibility to framing. Using genome-wide association data and the gene-based principal components regression method, we examined genetic variations of 26 genes on the pathways in 1317 Chinese Han participants. Consistent with previous studies, we found that the genetic variations of the SLC6A4 gene and the COMT gene were associated with the framing effect. More importantly, we demonstrated that the genetic variations of the aromatic-L-amino-acid decarboxylase (DDC) gene, which is involved in the synthesis of both dopamine and serotonin, contributed to individual differences in the susceptibility to framing. Our findings shed light on the understanding of the genetic basis of affective decision-making. PMID:28431168

A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine1

PubMed Central

Schmidlin, Laure; Poutaraud, Anne; Claudel, Patricia; Mestre, Pere; Prado, Emilce; Santos-Rosa, Maria; Wiedemann-Merdinoglu, Sabine; Karst, Francis; Merdinoglu, Didier; Hugueney, Philippe

2008-01-01

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment. PMID:18799660

Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome.

PubMed

Hall, K T; Kossowsky, J; Oberlander, T F; Kaptchuk, T J; Saul, J P; Wyller, V B; Fagermoen, E; Sulheim, D; Gjerstad, J; Winger, A; Mukamal, K J

2016-10-01

Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2500 fewer steps compared with placebo (Pinteraction=0.04). There were no differences between clonidine and placebo among patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (Pinteraction=0.003) and quality of life (Pinteraction=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

PubMed Central

Mei, Yu; Kernodle, Bliss M.; Hill, John H.

2016-01-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

Novel single nucleotide polymorphisms of the bovine methyltransferase 3b gene and their association with meat quality traits in beef cattle.

PubMed

Liu, X; Guo, X Y; Xu, X Z; Wu, M; Zhang, X; Li, Q; Ma, P P; Zhang, Y; Wang, C Y; Geng, F J; Qin, C H; Liu, L; Shi, W H; Wang, Y C; Yu, Y

2012-08-16

DNA methylation is essential for adipose deposition in mammals. We screened SNPs of the bovine DNA methyltransferase 3b (DNMT3b) gene in Snow Dragon beef, a commercial beef cattle population in China. Nine SNPs were found in the population and three of six novel SNPs were chosen for genotyping and analyzing a possible association with 16 meat quality traits. The frequencies of the alleles and genotypes of the three SNPs in Snow Dragon beef were similar to those in their terminal-paternal breed, Wagyu. Association analysis disclosed that SNP1 was not associated with any of the traits; SNP2 was significantly associated with lean meat color score and chuck short rib score, and SNP3 had a significant effect on dressing percentage and back-fat thickness in the beef population. The individuals with genotype GG for SNP2 had a 25.7% increase in lean meat color score and a 146% increase in chuck short rib score, compared with genotype AA. The cattle with genotype AG for SNP3 had 35.7 and 24% increases in dressing percentage and 28.8 and 29.2% increases in back-fat thickness, compared with genotypes GG and AA, respectively. Genotypic combination analysis revealed significant interactions between SNP1 and SNP2 and between SNP2 and SNP3 for the traits rib-eye area and live weight. We conclude that there is considerable evidence that DNMT3b is a determiner of beef quality traits.

Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation.

PubMed

Król, P; Igielski, R; Pollmann, S; Kępczyńska, E

2015-05-01

Methyl jasmonate (MeJA) was tested by seed treatment for its ability to protect tomato seedlings against fusarium wilt caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. lycopersici. Isolated from Solanum lycopersicon L. seeds, cv. Beta fungus was identified as F. oxysporum f.sp. lycopersici Race 3 fungus by using phytopathological and molecular methods. MeJA applied at 0.01, 0.1 and 1 mM reduced spore germination and mycelial growth in vitro. Soaking of tomato seeds in MeJA solution at 0.1 mM for 1 h significantly enhanced the resistance level against the tested fungus in tomato seedlings 4 weeks after inoculation. The extracts from leaves of 15-day-old seedlings obtained from previously MeJA soaked seeds had the ability to inhibit in vitro spore germination of tested fungus. In these seedlings a significant increase in the levels phenolic compounds such as salicylic acid (SA), kaempferol and quercetin was observed. Up-regulation of phenylalanine ammonia-lyase (PAL5) and benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) genes and down-regulation of the isochorysmate synthase (ICS) gene in response to exogenous MeJA application indicate that the phenylalanine ammonia-lyase (PAL), not the isochorismate (IC) pathway, is the primary route for SA production in tomato. Moreover, the increased accumulation of the flavonols quercetin and kaempferol appears closely related to the increase of PAL5, chalcone synthase (CHS) and flavonol synthase/flavanone 3-hydroxylase-like (FLS) genes. Elevated levels of salicylic acid in seedlings raised from MeJA-soaked seeds were simultaneously accompanied by a decrease of jasmonic acid, the precursor of MeJA, and an increase of 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid. The present results indicate that the priming of tomato seeds with 0.1mM MeJA before sowing enables the seedlings grown from these seeds to reduce the attack of the soil-borne fungal pathogen F. oxysporum f.sp. lycopersici

MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

PubMed Central

Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

2016-01-01

MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

Recruitment of DNA methyltransferase I to DNA repair sites.

PubMed

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M Cristina; Leonhardt, Heinrich

2005-06-21

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

PubMed

Krivega, Ivan; Byrnes, Colleen; de Vasconcellos, Jaira F; Lee, Y Terry; Kaushal, Megha; Dean, Ann; Miller, Jeffery L

2015-07-30

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Jian-Yuan; Liang, Lingli; Gu, Xiyao; Li, Zhisong; Wu, Shaogen; Sun, Linlin; Atianjoh, Fidelis E.; Feng, Jian; Mo, Kai; Jia, Shushan; Lutz, Brianna Marie; Bekker, Alex; Nestler, Eric J.; Tao, Yuan-Xiang

2017-01-01

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG. PMID:28270689

Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src.

PubMed

Ostareck-Lederer, Antje; Ostareck, Dirk H; Rucknagel, Karl P; Schierhorn, Angelika; Moritz, Bodo; Huttelmaier, Stefan; Flach, Nadine; Handoko, Lusy; Wahle, Elmar

2006-04-21

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.

Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

PubMed

Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

2015-12-01

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metadynamics Simulation Study on the Conformational Transformation of HhaI Methyltransferase: An Induced-Fit Base-Flipping Hypothesis

PubMed Central

Ye, Fei; Zhao, Dan; Chen, Shijie; Jiang, Ren-Wang; Jiang, Hualiang; Luo, Cheng

2014-01-01

DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an “induced-fit” process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators. PMID:25045662

Engineering Clostridium beijerinckii with the Cbei_4693 gene knockout for enhanced ferulic acid tolerance.

PubMed

Liu, Jun; Guo, Ting; Shen, Xiaoning; Xu, Jiahui; Wang, Junzhi; Wang, Yanyan; Liu, Dong; Niu, Huanqing; Liang, Lei; Ying, Hanjie

2016-07-10

A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

Biotin and Lipoic Acid: Synthesis, Attachment and Regulation

PubMed Central

Cronan, John E.

2014-01-01

Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses

Analysis of Oxidative Stress Status, Catalase and Catechol-O-Methyltransferase Polymorphisms in Egyptian Vitiligo Patients

PubMed Central

Mehaney, Dina A.; Darwish, Hebatallah A.; Hegazy, Rehab A.; Nooh, Mohammed M.; Tawdy, Amira M.; Gawdat, Heba I.; El-Sawalhi, Maha M.

2014-01-01

Vitiligo is the most common depigmentation disorder of the skin. Oxidative stress is implicated as one of the probable events involved in vitiligo pathogenesis possibly contributing to melanocyte destruction. Evidence indicates that certain genes including those involved in oxidative stress and melanin synthesis are crucial for development of vitiligo. This study evaluates the oxidative stress status, the role of catalase (CAT) and catechol-O-Methyltransferase (COMT) gene polymorphisms in the etiology of generalized vitiligo in Egyptians. Total antioxidant capacity (TAC) and malondialdehyde (MDA) levels as well as CAT exon 9 T/C and COMT 158 G/A polymorphisms were determined in 89 patients and 90 age and sex-matched controls. Our results showed significantly lower TAC along with higher MDA levels in vitiligo patients compared with controls. Meanwhile, genotype and allele distributions of CAT and COMT polymorphisms in cases were not significantly different from those of controls. Moreover, we found no association between both polymorphisms and vitiligo susceptibility. In conclusion, the enhanced oxidative stress with the lack of association between CAT and COMT polymorphisms and susceptibility to vitiligo in our patients suggest that mutations in other genes related to the oxidative pathway might contribute to the etiology of generalized vitiligo in Egyptian population. PMID:24915010

Expression analysis of fertilization/early embryogenesis-associated genes in Phalaenopsis orchids.

PubMed

Chen, Jhun-Chen; Wei, Miao-Ju; Fang, Su-Chiung

2016-10-02

One of the distinct reproductive programs in orchid species is pollination-triggered ovule development and megasporogenesis. During sexual reproduction, fertilization occurs days to months after pollination. The molecular mechanisms evolved to carry out this strategic reproductive program remain unclear. In the August issue of Plant Physiology 1 , we report comprehensive studies of comparative genome-wide gene expression in various reproductive tissues and the molecular events associated with developmental transitions unique to sexual reproduction of Phalaenopsis aphrodite. Transcriptional factors and signaling components whose expression is specifically enriched in interior ovary tissues when fertilization occurs and embryos start to develop have been identified. Here, we report verification of additional fertilization-associated genes, DOMAINS REARRANGED METHYLTRANSFERASE 1 (PaDRM1), CHROMOMETHYLTRANSFERASE 1 (PaCMT1), SU(VAR)3-9 RELATED PROTEIN 1 (PaSUVR1), INDOLE-3-ACETIC ACID inducible 30-like 1 (PaIAA30L1), and ETHYLENE INSENSITIVE 3-like 1 (PaEIN3L1), and discuss their potential roles in gametophyte development, epigenetic reprogramming, and hormone regulation during fertilization and establishment of embryo development in Phalaenopsis orchids.

Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids.

PubMed

Angelovici, Ruthie; Batushansky, Albert; Deason, Nicholas; Gonzalez-Jorge, Sabrina; Gore, Michael A; Fait, Aaron; DellaPenna, Dean

2017-01-01

Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed's nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants. © 2017 American Society of Plant Biologists. All Rights Reserved.

Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

PubMed Central

Li, Mimi; Peng, Bin; Guo, Haisong; Yan, Qinqin; Hang, Yueyu

2013-01-01

The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants. PMID:24358289

Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent1

PubMed Central

Scalliet, Gabriel; Lionnet, Claire; Le Bechec, Mickaël; Dutron, Laurence; Magnard, Jean-Louis; Baudino, Sylvie; Bergougnoux, Véronique; Jullien, Frédéric; Chambrier, Pierre; Vergne, Philippe; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe

2006-01-01

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses. PMID:16361520

A Biotin Biosynthesis Gene Restricted to Helicobacter

PubMed Central

Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

2016-01-01

In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

Sialic acid catabolism and transport gene clusters are lineage specific in Vibrio vulnificus.

PubMed

Lubin, Jean-Bernard; Kingston, Joseph J; Chowdhury, Nityananda; Boyd, E Fidelma

2012-05-01

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

PubMed

Wang, Fei; Wang, Yitong; Wang, Hui; Shao, Naimin; Chen, Yuanyuan; Cheng, Yiyun

2014-11-01

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

PubMed

Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

2015-01-01

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

PubMed

O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

2015-08-03

The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms. Copyright © 2015 Elsevier B.V. All rights reserved.

Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina

2017-08-15

Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

PubMed

Desriac, N; Postollec, F; Coroller, L; Sohier, D; Abee, T; den Besten, H M W

2013-10-01

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour. Copyright © 2013 Elsevier B.V. All rights reserved.

Gene expression differences in the methionine remethylation and transsulphuration pathways under methionine restriction and recovery with D,L-methionine or D,L-HMTBA in meat-type chickens.

PubMed

Aggrey, S E; González-Cerón, F; Rekaya, R; Mercier, Y

2018-02-01

This study examined the molecular mechanisms of methionine pathways in meat-type chickens where birds were provided with a diet deficient in methionine from 3 to 5 weeks of age. The birds on the deficient diet were then provided with a diet supplemented with either D,L-methionine or D,L-HMTBA from 5 to 7 weeks. The diet of the control birds was supplemented with L-methionine from hatch till 7 weeks of age. We studied the mRNA expression of methionine adenosyltransferase 1, alpha, methionine adenosyltransferase 1, beta, 5-methyltetrahydrofolate-homocysteine methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase, betaine-homocysteine S-methyltransferase, glycine N-methyltransferase, S-adenosyl-L-homocysteine hydrolase and cystathionine beta synthase genes in the liver, duodenum, Pectoralis (P.) major and the gastrocnemius muscle at 5 and 7 weeks. Feeding a diet deficient in dietary methionine affected body composition. Birds that were fed a methionine-deficient diet expressed genes that indicated that remethylation occurred via the one-carbon pathway in the liver and duodenum; however, in the P. major and the gastrocnemius muscles, gene expression levels suggested that homocysteine received methyl from both folate and betaine for remethylation. Birds who were switched from a methionine deficiency diet to one supplemented with either D,L-methionine or D,L-HMTBA showed a downregulation of all the genes studied in the liver. However, depending on the tissue or methionine form, either folate or betaine was elicited for remethylation. Thus, mRNA expressions show that genes in the remethylation and transsulphuration pathways were regulated according to tissue need, and there were some differences in the methionine form. © 2017 Blackwell Verlag GmbH.

Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

PubMed

Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

2018-04-01

Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

Genotype distribution of estrogen receptor-alpha, catechol-O-methyltransferase, and cytochrome P450 17 gene polymorphisms in Caucasian women with uterine leiomyomas.

PubMed

Denschlag, Dominik; Bentz, Eva-Katrin; Hefler, Lukas; Pietrowski, Detlef; Zeillinger, Robert; Tempfer, Clemens; Tong, Dan

2006-02-01

To evaluate the association between the presence of uterine leiomyomas and three functional single nucleotide polymorphisms (SNPs) of the estrogen receptor alpha (ESR1), catechol-O-methyltransferase (COMT), and cytochrom P450 17 (CYP17A) genes, which have been described to modify the estrogen metabolism. Prospective case control study. Academic research institution. One hundred thirty women with clinically and surgically diagnosed uterine leiomyomas and 139 population controls. Peripheral venous puncture. Polymerase chain reaction and pyrosequencing were performed to genotype women with respect to the ESR1 IVS1-397 T/C (PvuII), COMT G158A, and the CYP17A 34T-->C SNPs. Comparing women with uterine leiomyomas and controls, no statistically significant differences with respect to allele frequency and genotype distribution were ascertained for ESR1 IVS 1-397 T/C (PvuII) (P=0.9 and P=0.6, respectively), COMT G158A (P=0.3 and P=0.6, respectively), and CYP17A 34T-->C (P=0.1 and P=0.5, respectively). When all two-way interactions of investigated SNPs were ascertained, no significant interactions were observed. In a multivariate model, no SNP was significantly associated with leiomyomas. Carriage of the ESR1 IVS1-397 T/C (PvuII), COMT G158A, and the CYP17A 34T-->C SNPs is not associated with the susceptibility to uterine leiomyoma in a Caucasian population.

Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

PubMed

Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

2014-04-18

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB. Copyright © 2014 Elsevier Inc. All rights reserved.

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario

2006-08-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed,more » purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.« less

Influence of variation in the catechol-O-methyltransferase gene on the clinical outcome after lumbar spine surgery for one-level symptomatic disc disease: a report on 176 cases.

PubMed

Rut, Marcin; Machoy-Mokrzyńska, Anna; Ręcławowicz, Daniel; Słoniewski, Paweł; Kurzawski, Mateusz; Droździk, Marek; Safranow, Krzysztof; Morawska, Michalina; Białecka, Monika

2014-02-01

This study was aimed at the evaluation of the relationship between genetic polymorphisms of catechol-O-methyltransferase (COMT) (rs4680:A > G-Val158Met, rs6269:A > G, rs4633:C > T, rs4818:C > G) and pain sensitivity after lumbar discectomy. All patients had one-level symptomatic disc herniation from L3 to S1. The primary data recorded included visual analogue pain scales assessing back and leg pain, Oswestry Disability Questionnaire assessing quality of life and pain intensity, received/filled pre- and postoperatively. Each subject was genotyped for single-nucleotide polymorphism in the COMT gene. Clinical outcome was measured by difference between pre- and postoperative values and those results were analyzed with genetics findings. Pain intensity was associated with the COMT polymorphism. Carriers of rs6269 AA, rs4633 TT, rs4818 CC, and rs4680 AA genotypes were characterized by the lowest preoperative scores related to pain intensity and lower pain intensity at 1 year after the surgery. The rs4633 CC, rs4680 GG genotypes demonstrated significant clinical improvement in VASBACK score at 1 year after the surgery. Patients with COMT haplotype associated with low metabolic activity of enzyme (A_C_C_G) showed better clinical outcome measured by ODI score and VASBACK score 1 year after surgery. We did not observe any significant correlation between leg pain and single-nucleotide polymorphisms in the COMT gene. The results of our study indicate that polymorphism in the COMT gene may play an important role in the mechanism of pain perception, which may have a potential implication for clinical decision-making in the future.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber, Apostichopus japonicus (Selenka), during aestivation

NASA Astrophysics Data System (ADS)

Wang, Tianming; Yang, Hongsheng; Zhao, Huan; Chen, Muyan; Wang, Bing

2011-11-01

The sea cucumber, Apostichopus japonicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1, Methyl-CpG-binding domain protein 2), and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40). Similarly, we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria

USGS Publications Warehouse

Woodall, C.A.; Warner, K.L.; Oremland, R.S.; Murrell, J.C.; McDonald, I.R.

2001-01-01

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyl-transferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon.

PubMed

Kouzai, Yusuke; Kimura, Mamiko; Yamanaka, Yurie; Watanabe, Megumi; Matsui, Hidenori; Yamamoto, Mikihiro; Ichinose, Yuki; Toyoda, Kazuhiro; Onda, Yoshihiko; Mochida, Keiichi; Noutoshi, Yoshiteru

2016-03-02

Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. We propose that the Brachypodium immune

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

PubMed Central

Gopal-Srivastava, R; Mallonee, D H; White, W B; Hylemon, P B

1990-01-01

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium. Images PMID:2376563

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

PubMed

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Comparison of gene expression and fatty acid profiles in concentrate and forage finished beef.

PubMed

Buchanan, J W; Garmyn, A J; Hilton, G G; VanOverbeke, D L; Duan, Q; Beitz, D C; Mateescu, R G

2013-01-01

Fatty acid profiles and intramuscular expression of genes involved in fatty acid metabolism were characterized in concentrate- (CO) and forage- (FO) based finishing systems. Intramuscular samples from the adductor were taken at slaughter from 99 heifers finished on a CO diet and 58 heifers finished on a FO diet. Strip loins were obtained at fabrication to evaluate fatty acid profiles of LM muscle for all 157 heifers by using gas chromatography fatty acid methyl ester analysis. Composition was analyzed for differences by using the General Linear Model (GLM) procedure in SAS. Differences in fatty acid profile included a greater atherogenic index, greater percentage total MUFA, decreased omega-3 to omega-6 ratio, decreased percentage total PUFA, and decreased percentage omega-3 fatty acids in CO- compared with FO-finished heifers (P<0.05). Fatty acid profiles from intramuscular samples were ranked by the atherogenic index, and 20 heifers with either a high (HAI; n=10) or low (LAI; n=10) atherogenic index were selected for gene expression analysis using real-time PCR (RT-PCR). Gene expression data for the 20 individuals were analyzed as a 2 by 2 factorial arrangement of treatments using the GLM procedure in SAS. There was no significant diet × atherogenic index interaction identified for any gene (P>0.05). Upregulation was observed for PPARγ, fatty acid synthase (FASN), and fatty acid binding protein 4 (FABP4) in FO-finished compared with CO-finished heifers in both atherogenic index categories (P<0.05). Upregulation of diglyceride acyl transferase 2 (DGAT2) was observed in FO-finished heifers with a HAI (P<0.05). Expression of steroyl Co-A desaturase (SCD) was upregulated in CO-finished heifers with a LAI, and downregulated in FO-finished heifers with a HAI (P<0.05). Expression of adiponectin (ADIPOQ) was significantly downregulated in CO-finished heifers with a HAI compared with all other categories (P<0.05). The genes identified in this study which exhibit

Codon usage and amino acid usage influence genes expression level.

PubMed

Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

2018-02-01

Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

PubMed

Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria

PubMed Central

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S.; Bhatt, Apoorva

2012-01-01

Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. PMID:22520756

MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.

PubMed

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S; Bhatt, Apoorva

2012-04-20

Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. Copyright © 2012 Elsevier Ltd. All rights reserved.

Computational fishing of new DNA methyltransferase inhibitors from natural products.

PubMed

Maldonado-Rojas, Wilson; Olivero-Verbel, Jesus; Marrero-Ponce, Yovani

2015-07-01

DNA methyltransferase inhibitors (DNMTis) have become an alternative for cancer therapies. However, only two DNMTis have been approved as anticancer drugs, although with some restrictions. Natural products (NPs) are a promising source of drugs. In order to find NPs with novel chemotypes as DNMTis, 47 compounds with known activity against these enzymes were used to build a LDA-based QSAR model for active/inactive molecules (93% accuracy) based on molecular descriptors. This classifier was employed to identify potential DNMTis on 800 NPs from NatProd Collection. 447 selected compounds were docked on two human DNA methyltransferase (DNMT) structures (PDB codes: 3SWR and 2QRV) using AutoDock Vina and Surflex-Dock, prioritizing according to their score values, contact patterns at 4 Å and molecular diversity. Six consensus NPs were identified as virtual hits against DNMTs, including 9,10-dihydro-12-hydroxygambogic, phloridzin, 2',4'-dihydroxychalcone 4'-glucoside, daunorubicin, pyrromycin and centaurein. This method is an innovative computational strategy for identifying DNMTis, useful in the identification of potent and selective anticancer drugs. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

PubMed Central

Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

2015-01-01

ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

Nicotinamide N‐methyltransferase expression decreases in iron overload, exacerbating toxicity in mouse hepatocytes

PubMed Central

Koppe, Tiago; Patchen, Bonnie; Cheng, Aaron; Bhasin, Manoj; Vulpe, Chris; Schwartz, Robert E.; Moreno‐Navarrete, Jose Maria; Fernandez‐Real, Jose Manuel

2017-01-01

Iron overload causes the generation of reactive oxygen species that can lead to lasting damage to the liver and other organs. The goal of this study was to identify genes that modify the toxicity of iron overload. We studied the effect of iron overload on the hepatic transcriptional and metabolomic profile in mouse models using a dietary model of iron overload and a genetic model, the hemojuvelin knockout mouse. We then evaluated the correlation of nicotinamide N‐methyltransferase (NNMT) expression with body iron stores in human patients and the effect of NNMT knockdown on gene expression and viability in primary mouse hepatocytes. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism and that NNMT, an enzyme that methylates nicotinamide and regulates hepatic glucose and cholesterol metabolism, is one of the most strongly down‐regulated genes in the liver in both genetic and dietary iron overload. We found that hepatic NNMT expression is inversely correlated with serum ferritin levels and serum transferrin saturation in patients who are obese, suggesting that body iron stores regulate human liver NNMT expression. Furthermore, we demonstrated that adenoviral knockdown of NNMT in primary mouse hepatocytes exacerbates iron‐induced hepatocyte toxicity and increases expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while overexpression of NNMT partially reversed these effects. Conclusion: Iron overload alters glucose and nicotinamide transcriptional and metabolic pathways in mouse hepatocytes and decreases NNMT expression, while NNMT deficiency worsens the toxic effect of iron overload. For these reasons, NNMT may be a drug target for the prevention of iron‐induced hepatotoxicity. (Hepatology Communications 2017;1:803–815) PMID:29404495

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA

PubMed Central

Ranaei-Siadat, Ehsan; Fabret, Céline; Seijo, Bili; Dardel, Frédéric; Grosjean, Henri; Nonin-Lecomte, Sylvie

2013-01-01

In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5′ and 3′ ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m5UψC. In tRNAs of gram-negative bacteria, formation of m5U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m5U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m5U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m5U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m5U in RNA was performed by NMR analysis. PMID:23603891

Gene-diet interaction of a common FADS1 variant with marine polyunsaturated fatty acids for fatty acid composition in plasma and erythrocytes among men.

PubMed

Takkunen, Markus J; de Mello, Vanessa D; Schwab, Ursula S; Kuusisto, Johanna; Vaittinen, Maija; Ågren, Jyrki J; Laakso, Markku; Pihlajamäki, Jussi; Uusitupa, Matti I J

2016-02-01

Limited information exists on how the relationship between dietary intake of fat and fatty acids in erythrocytes and plasma is modulated by polymorphisms in the FADS gene cluster. We examined gene-diet interaction of total marine PUFA intake with a known gene encoding Δ-5 desaturase enzyme (FADS1) variant (rs174550) for fatty acids in erythrocyte membranes and plasma phospholipids (PL), cholesteryl esters (CE), and triglycerides (TG). In this cross-sectional study, fatty acid compositions were measured using GC, and total intake of polyunsaturated fat from fish and fish oil was estimated using a food frequency questionnaire in a subsample (n = 962) of the Metabolic Syndrome in Men Study. We found nominally significant gene-diet interactions for eicosapentaenoic acid (EPA, 20:5n-3) in erythrocytes (pinteraction = 0.032) and for EPA in plasma PL (pinteraction = 0.062), CE (pinteraction = 0.035), and TG (pinteraction = 0.035), as well as for docosapentaenoic acid (22:5n-3) in PL (pinteraction = 0.007). After excluding omega-3 supplement users, we found a significant gene-diet interaction for EPA in erythrocytes (pinteraction < 0.003). In a separate cohort of the Kuopio Obesity Surgery Study, the same locus was strongly associated with hepatic mRNA expression of FADS1 (p = 1.5 × 10(-10) ). FADS1 variants may modulate the relationship between marine fatty acid intake and circulating levels of long-chain omega-3 fatty acids. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transcriptional Profiling of Sorghum Induced by Methyl Jasmonate, Salicylic Acid, and Aminocyclopropane Carboxylic Acid Reveals Cooperative Regulation and Novel Gene Responses1[w

PubMed Central

Salzman, Ron A.; Brady, Jeff A.; Finlayson, Scott A.; Buchanan, Christina D.; Summer, Elizabeth J.; Sun, Feng; Klein, Patricia E.; Klein, Robert R.; Pratt, Lee H.; Cordonnier-Pratt, Marie-Michèle; Mullet, John E.

2005-01-01

We have conducted a large-scale study of gene expression in the C4 monocot sorghum (Sorghum bicolor) L. Moench cv BTx623 in response to the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene precursor aminocyclopropane carboxylic acid. Expression profiles were generated from seedling root and shoot tissue at 3 and 27 h, using a microarray containing 12,982 nonredundant elements. Data from 102 slides and quantitative reverse transcription-PCR data on mRNA abundance from 171 genes were collected and analyzed and are here made publicly available. Numerous gene clusters were identified in which expression was correlated with particular signaling compound and tissue combinations. Many genes previously implicated in defense responded to the treatments, including numerous pathogenesis-related genes and most members of the phenylpropanoid pathway, and several other genes that may represent novel activities or pathways. Genes of the octadecanoic acid pathway of jasmonic acid (JA) synthesis were induced by SA as well as by MeJA. The resulting hypothesis that increased SA could lead to increased endogenous JA production was confirmed by measurement of JA content. Comparison of responses to SA, MeJA, and combined SA+MeJA revealed patterns of one-way and mutual antagonisms, as well as synergistic effects on regulation of some genes. These experiments thus help further define the transcriptional results of cross talk between the SA and JA pathways and suggest that a subset of genes coregulated by SA and JA may comprise a uniquely evolved sector of plant signaling responsive cascades. PMID:15863699