Involvement of DNA methylation in the regulation of STS10 gene expression in Vitis amurensis

DNA methylation is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. However, it is not known whether DNA methylation is implicated in the genetic regulation of plant secondary metabolism, including resveratrol biosynthesis. Resveratrol is a naturally occurring polyphenol that is present in grapes, peanuts, and other plant sources, and it exhibits a wide range of valuable biologically active properties. The transformation of the wild-growing grape Vitis amurensis with the oncogene rolB from Agrobacterium rhizogenes has been demonstrated to considerably increase resveratrol production. To investigate whether DNA methylation regulates resveratrol biosynthesis, we treated both rolB transgenic and empty vector control V. amurensis cell cultures with the DNA demethylation agent 5-azacytosine (azaC). The azaC treatment significantly increased stilbene synthase 10 gene (VaSTS10) expression and resveratrol content in the V. amurensis cell cultures. Using bisulfite sequencing, we examined the methylation status of VaSTS10 in cell cultures under normal conditions and after azaC treatment. Both the promoter and 3′-end of the protein coding region of the VaSTS10 gene were hypermethylated (54–67 %) in the control cell culture. The rolB transgenic cell culture had high levels of resveratrol and lower hypermethylation levels of the VaSTS10 gene (20–47 %). The azaC treatment resulted in reduction in the DNA methylation levels in the promoter and coding regions of the VaSTS10 gene in both cell cultures. These data suggest that the DNA methylation may be involved in the control of resveratrol biosynthesis via the regulation of STS genes expression.     

Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis

DNA becomes methylated in vivo through the action of a specific group of enzymes known as methyltransferases or methylases. Plants are known to possess the methyltransferases (Met), chromo methyltransferases (CMT), and domainrearranged methyltransferases (DRM) methylase families, which affect cytosine methylation within different contexts. DNA methylation has been proposed to play a role in secondary plant metabolism, but there is a lack of valid data connecting these two processes. In this study, we treated control and transformed with rolB gene from Agrobacterium rhizogenes cell cultures of Vitis amurensis with the demethylation agent 5-azacytidine (azaC). The purpose of the current investigation was to study effects of induced DNA demethylation on methyltransferase gene expression in connection to resveratrol production, a naturally occurring polyphenol that has a wide range of intriguing biological properties. Using semi-quantitative and real-time PCR, we showed that rolB gene transformation of V. amurensis cells decreased Met and CMT expression, but significantly increased DRM expression. AzaC treatment of the control and the rolB-transgenic calli significantly increased expression of all methylases (excluding Met). Following 3 months of azaC treatment, we detected significantly elevated levels of rolB gene expression in the transgenic calli. In current paper, we discuss how methylase expression may influence resveratrol biosynthesis and rolB transgene expression. Effects of azaC application are discussed.     

Resveratrol content and expression patterns of stilbene synthase genes in <Emphasis Type="Italic">Vitis amurensis</Emphasis> cells treated with 5-azacytidine

DNA methylation is known to be involved in the regulation of plant development and defense mechanisms. However, there is a general lack of data on the role of methylation in plant secondary metabolism. We have investigated the effect of a cytidine analog, 5-azacytidine (azaC), which is known to block DNA methylation, on resveratrol biosynthesis and stilbene synthase (STS) gene expression in Vitis amurensis cultured cells. Resveratrol is a naturally occurring polyphenol that has been reported to exhibit a wide range of important biological and pharmacological properties. We previously obtained a control cell line of V. amurensis (VV) as well as a rolB-transgenic cell line of V. amurensis (VB2) that has a higher level of resveratrol accumulation. In our experimental setup, the azaC-treated VV and VB2 calli produced 0.092% and 0.455% dry weight (DW) resveratrol, respectively. We found that treatment with 200 μM of azaC resulted in 1.9- and 2.0-fold increases in resveratrol production in VV and VB2 calli, respectively. A quantitative real-time PCR assay for STS gene expression in the azaC-treated VV and VB2 cells revealed that there were statistically increased expression levels of VaSTS10 in VV calli and of VaSTS5, VaSTS6, and VaSTS10 in VB2 calli. These results demonstrate that azaC is able to increase resveratrol production in V. amurensis calli through a mechanism that involves the induction of STS gene expression.     

Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5′- and 3′-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5′- and 3′-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5′- and 3′- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.     

The asymmetric methylation of CG palindromic dinucleotides increases sister-chromatid exchanges

Treatment with the base analogue, 5azaC, increases SCEs in CHO but not in mosquito cells. On the other hand, both types of cells show equivalent increases in exchanges when treated with other compounds, such as mitomycin C. Vertebrate DNA is heavily methylated while diptera DNA is heavily demethylated. The sequence of events leading to an increase in SCEs in CHO cells is as follows: first of all, Cs are replaced by 5azaC; in the next cell cycle, CG palindromic dinucleotides exhibit an asymmetric configuration, the Cs in the parental DNA strand being methylated and the Cs in the daughter DNA strand demethylated; after one more cycle, half of the chromosomes show symmetric methylation and the other half symmetric demethylation of both Cs in CG palindromes. The increase of SCEs occurs in the second cell cycle when the hemimethylated DNA enters replication. DNA hemimethylation is believed to be an intermediate stage in the process of demethylation that accompanies gene expression. If so, gene demethylation would be a cause of SCE increase in normal vertebrate cells.     

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of calcium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02?% dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1–0.8?% DW). Treatment with the CI at a 1?μM concentration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10?μM significantly decreased resveratrol production by 2–4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1?μM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expression of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concentrations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.     

Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 ^R132) and IDH2 (IDH2 ^R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.     

Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca²⁺/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.     

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.     

Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.     

Culturing conditions highly affect DNA methylation and gene expression levels in MCF7 breast cancer cell line

The levels of DNA methylation and their role in gene expression are key factors that could affect diagnosis, prognosis, and treatment options of different diseases. In this study, the methylation levels of 22 genes that are mostly correlated to breast cancer were determined using EpiTect methyl II PCR array. This analysis was performed to determine the effect of cells’ passage number and the use of antibiotics in the culturing media on gene methylation levels in MCF7 cell line. DNA methylation levels of PTGS2, ADAM23, HIC1, and PYCARD were found to be significantly different among different passages. While the DNA methylation levels of CCNA1, RASSF1, and THBS1 were found to be affected by the use of 1% of penicillin/streptomycin in the culture media. Gene expression analysis after demethylation using 5-Aza-2′-deoxycytidine showed that the gene expression levels of the hypermethylated genes varied between different passage numbers. This study shows that the presence of antibiotic within cultured media and cell line’s passage number could greatly affect the methylation levels that need to be considered in future studies on cell lines.     

A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA

Summary A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M₁ progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M₂ progenies, obtained by self-fertilization of dwarf M₁ plants, were also dwarf, while those from tall M₁ plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m⁵C) content in comparison with DNA from untreated samples. A similar reduction in the m⁵C content was also observed in the M₁ and M₂ progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.