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5 Hydroxymethylcytosine

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Gerd P. Pfeifer – One of the best experts on this subject based on the ideXlab platform.

  • Single Base Resolution Analysis of 5-Methylcytosine and 5Hydroxymethylcytosine by RRBS and TAB-RRBS
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Maria A. Hahn, Gerd P. Pfeifer
    Abstract:

    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 5Hydroxymethylcytosine. 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 5Hydroxymethylcytosine. 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 5Hydroxymethylcytosine combined (RRBS) and for specific analysis of 5Hydroxymethylcytosine (TAB-RRBS).

  • 5Hydroxymethylcytosine: a stable or transient DNA modification?
    Genomics, 2014
    Co-Authors: Maria A. Hahn, Piroska E. Szabó, Gerd P. Pfeifer
    Abstract:

    The DNA base 5Hydroxymethylcytosine (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.

  • The role of 5Hydroxymethylcytosine in human cancer
    Cell and tissue research, 2014
    Co-Authors: Gerd P. Pfeifer, Wenying Xiong, Maria A. Hahn, Seung-gi Jin
    Abstract:

    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 5Hydroxymethylcytosine (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.

Chunxiao Song – One of the best experts on this subject based on the ideXlab platform.

  • Quantitation and mapping of the epigenetic marker 5Hydroxymethylcytosine.
    BioEssays : news and reviews in molecular cellular and developmental biology, 2017
    Co-Authors: Ying Qing, Chunxiao Song, Zhiqi Tian, Yongyao Wang, Jiangang Long, Jiajie Diao
    Abstract:

    We here review primary methods used in quantifying and mapping 5Hydroxymethylcytosine (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.

  • dynamics of 5 Hydroxymethylcytosine during mouse spermatogenesis
    Nature Communications, 2013
    Co-Authors: Shangying Liao, Chunxiao Song, Min Wang, Chuan He, Fuchou Tang
    Abstract:

    Changes in DNA methylation during mammalian spermatogenesis are poorly understood. The authors show that the content of 5Hydroxymethylcytosine, a stable intermediate of DNA demethylation, changes dynamically during mouse spermatogenesis and is associated with functional genomic regions and transcription.

  • Tet-assisted bisulfite sequencing of 5Hydroxymethylcytosine
    Nature protocols, 2012
    Co-Authors: Gary C. Hon, Chunxiao Song, Keith E. Szulwach, Peng Jin, Bing Ren
    Abstract:

    A complete understanding of the potential function of 5Hydroxymethylcytosine (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.

Maria A. Hahn – One of the best experts on this subject based on the ideXlab platform.

  • Single Base Resolution Analysis of 5-Methylcytosine and 5Hydroxymethylcytosine by RRBS and TAB-RRBS
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Maria A. Hahn, Gerd P. Pfeifer
    Abstract:

    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 5Hydroxymethylcytosine. 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 5Hydroxymethylcytosine. 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 5Hydroxymethylcytosine combined (RRBS) and for specific analysis of 5Hydroxymethylcytosine (TAB-RRBS).

  • 5Hydroxymethylcytosine: a stable or transient DNA modification?
    Genomics, 2014
    Co-Authors: Maria A. Hahn, Piroska E. Szabó, Gerd P. Pfeifer
    Abstract:

    The DNA base 5Hydroxymethylcytosine (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.

  • The role of 5Hydroxymethylcytosine in human cancer
    Cell and tissue research, 2014
    Co-Authors: Gerd P. Pfeifer, Wenying Xiong, Maria A. Hahn, Seung-gi Jin
    Abstract:

    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 5Hydroxymethylcytosine (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.