Down-regulation of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-homocysteine hydrolase reveals a role of cytokinin in promoting transmethylation reactions

S-adenosyl-L: -homocysteine hydrolase (SAHH) is a key enzyme for maintenance of cellular transmethylation potential. Although a cytokinin-binding activity had been hypothesized for SAHH, the relation between cytokinin and transmethylation reactions has not been elucidated. Here we show that, of the two Arabidopsis thaliana SAHH genes, AtSAHH1 has a much higher expression level than AtSAHH2. A T-DNA insertion mutant of AtSAHH1 (sahh1-1) and the RNA interference (RNAi) plants (dsAtSAHH2) accumulated a higher level of cytokinins, exhibited phenotypic changes similar to those of cytokinin-overproducers, and their global DNA methylation status was reduced. On the other hand, cytokinins positively regulate the transmethylation pathway genes, including AtSAHH1, AtADK1 (for adenosine kinase), and this regulation involves the cytokinin activity. Furthermore, expression of three cytosine DNA methyltransferase genes examined was inducible by cytokinin treatment. Unlike adenine and adenosine which are SAHH inhibitors, the adenine-type cytokinins have no effect on SAHH activity at protein level. Changing of endogenous cytokinin levels by transgene expression resulted in alterations of DNA methylation status in the sahh1-1 background, suggesting that cytokinins promote DNA methylation, at least under transmethylation stringent conditions. These data demonstrate that the phytohormone cytokinin plays a role in promoting transmethylation reactions, including DNA methylation.     

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.     

Homocysteine-mediated expression of SAHH, DNMTs, MBD2, and DNA hypomethylation potential pathogenic mechanism in VSMCs

Homocysteine (Hcy) is a well-established risk factor for atherosclerosis and may cause dysregulation of gene expression, but the characteristics and the key links involved in its pathogenic mechanisms are still poorly understood. The aim of this study was to explore (i) the effects of Hcy on DNA methylation in vascular smooth muscle cells (VSMCs) and (ii) the underlying mechanism of Hcy-induced changes in DNA methylation patterns in relation to atherosclerosis. We examined the levels of gDNA methylation, namely, the Alu and line-1 element sequences, which can serve as a surrogate marker for gDNA methylation, and also investigated the effects of Hcy on the intracellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations as well as the expressions of SAH hydrolase (SAHH), DNA methyltransferase3a (DNMT3a), DNMT3b, and methyl-CpG-binding domain 2 (MBD2). We found that clinically relevant levels of Hcy (0-500 microM) induced elevation of SAH, declination of SAM and SAM/SAH ratio, and reduction in expression of SAHH and MBD2, but increased the activity of DNMT3a and DNMT3b compared to the control group (p < 0.05). We found also that the genome-wide hypomethylation is a common feature of gDNA in the VSMCs cultured with Hcy. In conclusion, these results suggest that Hcy-induced DNA methylation may be an important potential pathogenic mechanism in the development of atherosclerosis, and may become a therapeutic target for preventing Hcy-induced atherosclerosis.     

Cytoplasmic protein methylation is essential for neural crest migration

As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration.     

Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue

Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.     

Sustaining S-adenosyl-l-methionine-dependent methyltransferase activity in plant cells

Many biochemical reactions in plants involve the transfer of a methyl group from S -adenosyl- l -methionine (SAM). The transfer of the methyl group from SAM generates S -adenosyl- l -homocysteine (SAH), a potent inhibitor of SAM-dependent methyltransferases (MTs). To mitigate the toxic effects of SAH on MT activity, SAH is removed by SAH hydrolase (SAHH, EC 3.3.1.1) in a reaction generating homocysteine and adenosine (Ado). However, SAHH catalyzes a reversible reaction that is favored to move in the direction of SAH hydrolysis only by removal of these products. Removal of Ado is reported to exert a greater influence on promoting SAH hydrolysis. Whereas animals appear to rely upon Ado deaminase (EC 3.5.4.4) to catabolize Ado, plants appear to use adenosine kinase (EC 2.7.1.20) for this important role. Compounds undergoing methylation represent a broad spectrum of chemically diverse substrates ranging from nucleic acids, lipids and cell wall components to comparatively simpler amines, alcohols and metal halides. Given the diverse nature of methyl acceptor compounds, it is very likely that the demand for SAM synthesis and SAH removal changes both temporally and spatially during the course of plant growth and development. Plants also use SAM as a precursor for the synthesis of ethylene, polyamines, biotin and nicotianamine. These uses are also expected to undergo changes reflective of the metabolic activities of different plants, plant organs, or cells. This review examines the various uses of SAM in plants and addresses how they allocate this resource to satisfy potentially competing needs.     

In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T₂ progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.     

NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation

The role of copper in biological phenomena that involve signal transduction is poorly understood. A well-defined cellular model of neuronal differentiation has been utilized to examine the requirement for copper during nerve growth factor (NGF) signal transduction that results in neurite outgrowth. Experiments demonstrate that NGF increases cellular copper content within 3 days of treatment. Copper chelators reduce the effects of NGF on neurite outgrowth and copper accumulation. The effects of tetraethylene pentamine (TEPA), a copper-specific chelator, are reversible by removal from the culture medium and/or by addition of equimolar copper chloride. Because previous work demonstrated that NGF increases protein methylation in PC12 cells, we examined whether TEPA also inhibits S-adenosylhomocysteine hydrolase (SAHH), an essential copper enzyme involved in all protein methylation reactions. In addition to direct in vitro inhibition of SAHH, we show that TEPA decreases protein arginine methyltransferase 1(PRMT1)-specific enzyme activity in PC12 cells and sympathetic neurons. These data comprise the first biochemical and cellular evidence to address the mechanism of copper involvement in neuronal differentiation.     

Structure,evolution, and inhibitor interaction of S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum

S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s).     

Dual methylation pathways in lignin biosynthesis

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.     

Long non-coding RNA H19/SAHH axis epigenetically regulates odontogenic differentiation of human dental pulp stem cells

Long noncoding RNAs (lncRNAs) are emerging as important regulators in molecular processes and may play vital roles in odontogenic differentiation of human dental pulp stem cells (hDPSCs). However, their functions remain to be elucidated. As lncRNA H19 is one of the most classical lncRNA, which plays essential roles in cellular differentiation, thus we explored the effects and mechanisms of H19 in odontogenic differentiation of hDPSCs. Stable overexpression and knockdown of H19 in hDPSCs were constructed using recombinant lentiviruses containing H19 and short hairpin-H19 expression cassettes, respectively. Alkaline phosphatase (ALP) assay, Alizarin red staining assay, von kossa staining, quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescent staining results indicated that overexpression of H19 in hDPSCs positively regulates the odontogenic differentiation of hDPSCs, while knockdown of H19 in hDPSCs inhibits odontogenic differentiation of hDPSCs. Further, we found that H19 promotes the odontogenic differentiation of hDPSCs through S-adenosylhomocysteine hydrolase (SAHH) epigenetically regulates the methylation and expression of distal-less homeobox (DLX3) gene. Herein, for the first time, we determined that H19/SAHH axis epigentically regulates odontogenic differentiaiton of hDPSCs by inhibiting the DNA methyltransferase 3B (DNMT3B)-mediated methylation of DLX3. Our findings provide a new insight into how H19/SAHH axis play its role in odontogenic differentiation of hDPSCs, and would be helpful in developing therapeutic approaches for dentin regeneration based on stem cells.     

The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression

Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5' and 3' ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants.