The "6th base" in DNA

5-Hydroxymethylcytosine (5-hmC) research tools

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The so-called "6th base" 5-hydroxymethylcytosine (5-hmC) is a DNA modification that is an important epigenetic marker associated with e.g. stem cell differentiation and brain development[1].

Its biological role however, is not yet fully understood especially with regard to the oxidation cascade 5‑mC →  5‑hmC → 5‑fC → 5‑caC catalyzed by the Tet family of cytosine oxygenases[2-4].

Check out our epigenetic 5-hmC research tools:

  Preparation of 5-hmC DNA fragments

5-hydroxymethylated DNA fragments can be used as sequencing control[5-9] or for pull-down of 5-hmC-binding proteins from cellular lysate[10]. They are typically generated by enzymatic incorporation of 5-hmdCTP (Fig. 1):

Figure 1: Structure of 5-hmdCTP.

  • Purity: ≥ 95 % (HPLC), < 0.5 % dCTP (HPLC)
  • Functionality: Incorporation into DNA by PCR with Taq polymerase (100 % dCTP substitution)
  • Fragment size: amplification of templates up to 1500 bp tested

  5-hmC detection in genomic DNA

The discrimination of 5-hmC from its analog 5-mC is essential to determine the modification's influence on gene regulation. Traditional sodium bisulfite sequencing however, does not distinguish between these two[11]. Song et al.[12] reported an approach for the selective detection of 5-hmC residues in genomic DNA based on UDP-6-azide-glucose (UDP-6-N3-Glc) (Fig. 2).

Figure 2: Structure of UDP-6-N3-Glc.

  Selected References

[1] Branco et al. (2012) Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nature Reviews Genetics 13:7.
[2] Tahiliani et al. (2009) Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1. Science 324 (5929):930.
[3] Ito et al. (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333 (6047):1300.
[4] He et al. (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333 (6047):1303.
[5] Booth et al. (2014) Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat. Chem. 6 (5):435.
[6] Booth et al. (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8 (10):1841.
[7] Yu et al. (2012) Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat. Protoc. 7 (12):2159.
[8] Szwagierczak et al. (2011) Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine. Nucl. Acids Res. 39 (12):5149.
[9] Szwagierczak et al. (2010) Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucl. Acids Res. 38 (19):e181.
[10] Lafaye et al. (2014) DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation. Biochem. Biophys. Res. Comm. 446:341.
[11] Huang et al. (2010) The behaviour of 5-hydroxymethycytosine in bisulfite sequencing. PLOS One 5 (1):e8888.
[12] Song et al. (2011) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nature Biotech 29 (1):68.
[13] Li et al. (2012) Selective Capture of 5-hydroxymethylcytosine from Genomic DNA. J. Vis. Exp. 68:e4441.
[14] Song et al. (2016) Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation. PNAS 113 (16):4339.

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