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Gerd P. Pfeifer - One of the best experts on this subject based on the ideXlab platform.
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Single Base Resolution Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine by RRBS and TAB-RRBS
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Maria A. Hahn, Gerd P. PfeiferAbstract:Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-Hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-Hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5'CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-Hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-Hydroxymethylcytosine (TAB-RRBS).
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5-Hydroxymethylcytosine: a stable or transient DNA modification?
Genomics, 2014Co-Authors: Maria A. Hahn, Piroska E. Szabó, Gerd P. PfeiferAbstract:The DNA base 5-Hydroxymethylcytosine (5hmC) is produced by enzymatic oxidation of 5-methylcytosine (5mC) by 5mC oxidases (the Tet proteins). Since 5hmC is recognized poorly by DNA methyltransferases, DNA methylation may be lost at 5hmC sites during DNA replication. In addition, 5hmC can be oxidized further by Tet proteins and converted to 5-formylcytosine and 5-carboxylcytosine, two bases that can be removed from DNA by base excision repair. The completed pathway represents a replication-independent DNA demethylation cycle. However, the DNA base 5hmC is also known to be rather stable and occurs at substantial levels, for example in the brain, suggesting that it represents an epigenetic mark by itself that may regulate chromatin structure and transcription. Focusing on a few well-studied tissues and developmental stages, we discuss the opposing views of 5hmC as a transient intermediate in DNA demethylation and as a modified DNA base with an instructive role.
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The role of 5-Hydroxymethylcytosine in human cancer
Cell and tissue research, 2014Co-Authors: Gerd P. Pfeifer, Wenying Xiong, Maria A. Hahn, Seung-gi JinAbstract:The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-Hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.
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Dynamics of 5-Hydroxymethylcytosine and Chromatin Marks in Mammalian Neurogenesis
Cell reports, 2013Co-Authors: Maria A. Hahn, Seung-gi Jin, Runxiang Qiu, Heying Zhang, Jun Wang, Jonathan Jui, Yong Jiang, Gerd P. PfeiferAbstract:DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-Hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.
Chunxiao Song - One of the best experts on this subject based on the ideXlab platform.
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Quantitation and mapping of the epigenetic marker 5-Hydroxymethylcytosine.
BioEssays : news and reviews in molecular cellular and developmental biology, 2017Co-Authors: Ying Qing, Chunxiao Song, Zhiqi Tian, Yongyao Wang, Jiangang Long, Jiajie DiaoAbstract:We here review primary methods used in quantifying and mapping 5-Hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.
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dynamics of 5 Hydroxymethylcytosine during mouse spermatogenesis
Nature Communications, 2013Co-Authors: Shangying Liao, Chuan He, Chunxiao Song, Min Wang, Fuchou TangAbstract:Changes in DNA methylation during mammalian spermatogenesis are poorly understood. The authors show that the content of 5-Hydroxymethylcytosine, a stable intermediate of DNA demethylation, changes dynamically during mouse spermatogenesis and is associated with functional genomic regions and transcription.
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Tet-assisted bisulfite sequencing of 5-Hydroxymethylcytosine
Nature protocols, 2012Co-Authors: Gary C. Hon, Chunxiao Song, Keith E. Szulwach, Peng Jin, Bing RenAbstract:A complete understanding of the potential function of 5-Hydroxymethylcytosine (5-hmC), a DNA cytosine modification in mammalian cells, requires an accurate single-base resolution sequencing method. Here we describe a modified bisulfite-sequencing method, Tet-assisted bisulfite sequencing (TAB-seq), which can identify 5-hmC at single-base resolution, as well as determine its abundance at each modification site. This protocol involves β-glucosyltransferase (β-GT)-mediated protection of 5-hmC (glucosylation) and recombinant mouse Tet1(mTet1)-mediated oxidation of 5-methylcytosine (5-mC) to 5-carboxylcytosine (5-caC). After the subsequent bisulfite treatment and PCR amplification, both cytosine and 5-caC (derived from 5-mC) are converted to thymine (T), whereas 5-hmC reads as C. The treated genomic DNA is suitable for both whole-genome and locus-specific sequencing. The entire procedure (which does not include data analysis) can be completed in 14 d for whole-genome sequencing or 7 d for locus-specific sequencing.
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5-Hydroxymethylcytosine (5-hmC) Specific Enrichment.
Bio-protocol, 2012Co-Authors: Keith E. Szulwach, Chunxiao Song, Peng JinAbstract:[Abstract] 5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.
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Sensitive and specific single-molecule sequencing of 5-Hydroxymethylcytosine
Nature methods, 2011Co-Authors: Chunxiao Song, Tyson A. Clark, Andrey Kislyuk, Qing Dai, Stephen Turner, Jonas KorlachAbstract:The DNA modification 5-Hydroxymethylcytosine has recently been implicated in several biological processes. Enrichment by selective chemical labeling in combination with single-molecule, real-time sequencing provides sensitive detection of this epigenetic mark in genomic DNA at base-pair resolution.
Maria A. Hahn - One of the best experts on this subject based on the ideXlab platform.
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Single Base Resolution Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine by RRBS and TAB-RRBS
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Maria A. Hahn, Gerd P. PfeiferAbstract:Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-Hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-Hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5'CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-Hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-Hydroxymethylcytosine (TAB-RRBS).
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5-Hydroxymethylcytosine: a stable or transient DNA modification?
Genomics, 2014Co-Authors: Maria A. Hahn, Piroska E. Szabó, Gerd P. PfeiferAbstract:The DNA base 5-Hydroxymethylcytosine (5hmC) is produced by enzymatic oxidation of 5-methylcytosine (5mC) by 5mC oxidases (the Tet proteins). Since 5hmC is recognized poorly by DNA methyltransferases, DNA methylation may be lost at 5hmC sites during DNA replication. In addition, 5hmC can be oxidized further by Tet proteins and converted to 5-formylcytosine and 5-carboxylcytosine, two bases that can be removed from DNA by base excision repair. The completed pathway represents a replication-independent DNA demethylation cycle. However, the DNA base 5hmC is also known to be rather stable and occurs at substantial levels, for example in the brain, suggesting that it represents an epigenetic mark by itself that may regulate chromatin structure and transcription. Focusing on a few well-studied tissues and developmental stages, we discuss the opposing views of 5hmC as a transient intermediate in DNA demethylation and as a modified DNA base with an instructive role.
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The role of 5-Hydroxymethylcytosine in human cancer
Cell and tissue research, 2014Co-Authors: Gerd P. Pfeifer, Wenying Xiong, Maria A. Hahn, Seung-gi JinAbstract:The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-Hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.
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Dynamics of 5-Hydroxymethylcytosine and Chromatin Marks in Mammalian Neurogenesis
Cell reports, 2013Co-Authors: Maria A. Hahn, Seung-gi Jin, Runxiang Qiu, Heying Zhang, Jun Wang, Jonathan Jui, Yong Jiang, Gerd P. PfeiferAbstract:DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-Hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.
Magnus Ingelman-sundberg - One of the best experts on this subject based on the ideXlab platform.
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Single base resolution analysis of 5-Hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.
Nucleic acids research, 2016Co-Authors: Maxim Ivanov, Mart Kals, Isabel Barragan, Lili Milani, Volker M. Lauschke, Philip Ewels, Max Käller, Tomas Axelsson, Janne Lehtiö, Magnus Ingelman-sundbergAbstract:To improve the epigenomic analysis of tissues rich in 5-Hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both h ...
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Ontogeny, distribution and potential roles of 5-Hydroxymethylcytosine in human liver function.
Genome biology, 2013Co-Authors: Maxim Ivanov, Mart Kals, Marina Kacevska, Isabel Barragan, Kie Kasuga, Anders Rane, Andres Metspalu, Lili Milani, Magnus Ingelman-sundbergAbstract:Background Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-Hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression.
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Ontogeny, distribution and potential roles of 5-Hydroxymethylcytosine in human liver function
Genome Biology, 2013Co-Authors: Maxim Ivanov, Mart Kals, Marina Kacevska, Isabel Barragan, Kie Kasuga, Anders Rane, Andres Metspalu, Lili Milani, Magnus Ingelman-sundbergAbstract:Background Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-Hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression. Results In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development. Conclusions Our findings suggest that 5-Hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.
Yuval Ebenstein - One of the best experts on this subject based on the ideXlab platform.
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Simple and cost-effective fluorescent labeling of 5-Hydroxymethylcytosine.
Methods and applications in fluorescence, 2016Co-Authors: Tamar Shahal, Ori Green, Uri Hananel, Yael Michaeli, Doron Shabat, Yuval EbensteinAbstract:The nucleobase 5-Hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation. The fluorescence intensity of the labeled sample is directly proportional to its 5-hmC content. We show that this simple and cost-effective technique is suitable for quantification of 5-hmC content in different mouse tissues.
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Spectroscopic Quantification of 5-Hydroxymethylcytosine in Genomic DNA
Analytical chemistry, 2014Co-Authors: Tamar Shahal, Yael Michaeli, Doron Shabat, Noa Gilat, Orit Redy-keisar, Yuval EbensteinAbstract:5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a “click reaction” with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement wit...