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Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases a...

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Autores principales: Choi, Woo Lee, Mok, Young Geun, Huh, Jin Hoe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865733/
https://www.ncbi.nlm.nih.gov/pubmed/33499041
http://dx.doi.org/10.3390/ijms22031072
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author Choi, Woo Lee
Mok, Young Geun
Huh, Jin Hoe
author_facet Choi, Woo Lee
Mok, Young Geun
Huh, Jin Hoe
author_sort Choi, Woo Lee
collection PubMed
description In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.
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spelling pubmed-78657332021-02-07 Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation Choi, Woo Lee Mok, Young Geun Huh, Jin Hoe Int J Mol Sci Article In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion. MDPI 2021-01-22 /pmc/articles/PMC7865733/ /pubmed/33499041 http://dx.doi.org/10.3390/ijms22031072 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Choi, Woo Lee
Mok, Young Geun
Huh, Jin Hoe
Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title_full Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title_fullStr Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title_full_unstemmed Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title_short Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation
title_sort application of 5-methylcytosine dna glycosylase to the quantitative analysis of dna methylation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865733/
https://www.ncbi.nlm.nih.gov/pubmed/33499041
http://dx.doi.org/10.3390/ijms22031072
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