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A Comparative Analysis of 5-Azacytidine- and Zebularine-Induced DNA Demethylation.

ABSTRACT: The nonmethylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of upregulated genes, with an overrepresentation of transposable element genes, in particular CACTA-like elements. This demonstrates that chemical demethylating agents have a disproportionately large effect on loci that are otherwise silenced by DNA methylation.

SUBMITTER: Griffin PT

PROVIDER: S-EPMC5015934 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A Comparative Analysis of 5-Azacytidine- and Zebularine-Induced DNA Demethylation.

Griffin Patrick T PT Niederhuth Chad E CE Schmitz Robert J RJ

G3 (Bethesda, Md.) 20160908 9

The nonmethylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had ...[more]

PMID: 27402357

Similar Datasets

A comparative analysis of 5-azacytidine and zebularine induced DNA demethylation using whole-genome bisulfite sequencing

Project description:The non-methylable cytosine analogs, 5-azacytidine and zebularine, are widely used to disrupt DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that 5-azacytidine and zebularine-treated seedlings have nearly indistinguishable patterns of DNA methylation genome-wide and that 5-azacytidine, despite being more unstable in aqueous solution, has a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of up-regulated genes and transposable element genes, particularly CACTA-like elements, demonstrating that chemical demethylating agents have a disproportionately large effect on loci that are silenced by DNA methylation.

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A comparative analysis of 5-azacytidine and zebularine induced DNA demethylation using whole-genome bisulfite sequencing

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Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

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Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

Project description:Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

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Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine.

Project description:In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.

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Zebularine, a DNA Methylation Inhibitor, Activates Anthocyanin Accumulation in Grapevine Cells.

Project description:Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated.

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