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Epigenetics & Chromatin

5-Hydroxymethylcytosine

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5-Hydroxymethylcytosine (5-hmC) Background

5-Hydroxymethylcytosine is a DNA methylation modification that is believed to occur as a result of enzymatic oxidation of 5-methylcytosine (5-mC) by the TET family of iron-dependent deoxygenases1. 5-Hydroxymethylcytosine can be found in elevated amounts in certain mammalian tissues, such as mouse Purkinje and granule neurons2. Alternatively, 5-hmC may be produced by the addition of formaldehyde to DNA cytosines by DNMT proteins3.

While a variety of techniques exist to study DNA methylation, most of these techniques were developed to distinguish 5-methylcytosine from normal cytosine. An evaluation of these same methods in the context of 5-hydroxymethylcytosine reveal that many are unable to be used for the analysis of 5-hmC. Bisulfite sequencing, for example, cannot distinguish between 5-mC and 5-hmC, just as methyl-binding protein methods (e.g. methylated CpG island recovery assay, MIRA) will only recognize 5-mC DNA4. To better understand the function of 5-hydroxymethylcytosine in the mammalian genome, new tools and techniques need to be employed.

5-Hydroxymethylcytosine Antibody Immunoprecipitation

DNA immunoprecipitation using a methylation specific antibody is one of the tools that can be used to differentiate 5-hmC and 5-mC DNA. During the immunoprecipitation, an antibody specific for the 5-hydroxymethylcytosine residue is used to bind DNA fragments containing this methylation mark. The antibody/DNA complex is then pulled down from the rest of the genomic DNA, resulting in a final elution that is highly enriched for 5-hydroxymethylcytosine DNA. Active Motif's hMeDIP Kit is designed for this exact purpose and has been optimized for use with double-stranded DNA.

5-Hydroxymethylcytosine Enzymes

Methylation-sensitive restriction enzymes are another way to discriminate between 5-hmC and 5-mC. To date, there is only one enzyme able to directly differentiate the two forms of DNA methylation, and that is the PvuRts1I restriction enzyme. The PvuRts1I enzyme is specific to 5-hmC DNA and will not digest 5-methylcytosine residues or unmethylated DNA5.

Another way to distinguish 5-mC and 5-hmC methylation is to modify the 5-hydroxymethylcytosine residue. The β-Glucosyltransferase enzyme is able to transfer a glucose moiety from uridine diphosphoglucose (UDP-Glucose) to the 5-hydroxymethylcytosine residue in double-stranded DNA, creating glucosyl-5-hmC DNA. Glucosylated DNA can be quantified or used to differentiate 5-hmC and 5-mC DNA using glucosyl-sensitive restriction enzymes6,7.

5-Hydroxymethylcytosine Biotin-based Enrichment

By utilizing the properties of the β-Glucosyltransferase enzyme, Active Motif has developed the Hydroxymethyl Collector™ Kit, which uses a biotin-based capture system to detect and enrich for DNA fragments containing 5-hydroxymethylcytosine. The Hydroxymethyl Collector Kit uses the β-Glucosyltransferase enzyme to transfer a modified glucose moiety to 5-hmC residues in double-stranded DNA.  A biotin conjugate is then chemically introduced onto the modified glucose, enabling enrichment with streptavidin magnetic beads7. This covalent labeling of 5-hmC ensures accurate capture of DNA fragments containing 5-hydroxymethylcytosine and is an efficient method to separate hydroxymethylation from traditional methylation.

5-Hydroxymethylcytosine Control DNA

Active Motif has also developed our Methylated DNA Standard Kit for use as a positive control when studying DNA methylation. The Methylated DNA Standard Kit contains three recombinant standards derived from the APC gene promoter. Each standard is 338 base pairs and contains 122 cytosine residues. One standard is completely unmethylated, while another standard is fully 5-methylcytosine methylated and the last standard is fully 5-hydroxymethylcytosine methylated. PCR primers are included with the Methylated DNA Standard Kit.

 

References

1. Tahiliani, M, et al. Science 324, 930-935 (2009).
2. Kriaucionis, S. and Heintz, N. Science 324, 929-930 (2009).
3. Liutkeviciute, Z. et al. Nat. Chem. Biol. 5, 400-402 (2009).
4. Jin, S.G., et al. Nucleic Acids Res 38,  (2010) doi:10.1093/nar/gkq223.
5. Szwagierczak, A. et al. Nucleic Acids Res (2011) doi:10.1093/nar/gkr118.
6. Szwagierczak, A. et al. Nucleic Acids Res (2010) doi:10.1093/nar/gkq684.
7. Song, C.X et al. Nature Biotechnology. 29, 68-72 (2011).