5-Hydroxymethylcytosine (5-hmC): an emerging player in DNA methylation

Localization, function and analysis of the sixth base. 

Cytosine is a molecule that has left a big mark in the field of epigenetics; with research into it revealing a complex role in the regulation of gene expression.

5-Hydroxymethylcytosine (5-hmC) has recently emerged as an epigenetic mark. Despite its initial discovery in bacteriophages in 1952 (Wyatt & Cohen, 1952), it was only reported in mammals in the 1970s (Penn et al., 1972). However, researchers failed to replicate these findings in later studies (Kothari & Shankar, 1976) and it took more than 30 years to confirm the existence of 5-hmC and gain insights into its molecular function. 

Progress was made in 2009 when, while assaying for 5-mC, researchers identified 5-hmC in human and mouse brains (Kriaucionis & Heintz, 2009). At the same time, another research group independently observed 5-hmC to be abundant in embryonic stem cells (Tahiliani et al., 2009).

These two high-impact Science publications, led to a revolution in epigenetics, with 5-hmC successfully staging a coup for the top methylation mark of interest.

Just as most researchers were beginning to realize that molecular inheritance was a process much more complex than simply four base pairs, a sixth base pair had emerged.

5-hmC localization

Although initially observed in neurons and embryonic stem cells, 5-hmC has since been extensively characterized for tissue specificity. 

High levels are often found in the central nervous system and the spinal cord (Globisch et al., 2010), while significantly lower levels are observed elsewhere. 5-hmC truly does appear to be a molecular mark of the nervous system.

5-hmC function

5-hmC was initially believed to be a by-product of oxidative stress, rather than a functional epigenetic mark. However, researchers observed an absence of other markers for oxidative stress in the presence of high 5-hmC levels.

It was then shown that 5-hmC is the product of DNA methylation, an active process involving the TET family of enzymes. Now, recent research suggests that not only do the intermediates of this reaction (5-hmC, 5-fC and 5-caC) have a role in the demethylation of DNA, but they may also act as epigenetic signals on their own (Iurlaro, et al., 2013).​

However, it appears that 5-hmC's true functional potential is only beginning to be realized. This is exemplified in the case of active demethylation, which occurs in post-mitotic adult neurons (Gavin et al., 2013).

Furthermore, recent research has also shown that there is a global loss of 5-hmC in cancerous cells of a number of non-brain tissues (Pfeifer et al., 2013), suggesting a potential role in cellular growth regulation.

5-hmC, 5-fC, 5-caC and the TET oxidative pathway

5-hmC is a key intermediate in cytosine demethylation as it can either be passively depleted through DNA replication or actively reverted to cytosine through oxidation reactions and base excision repair (Kohli & Zhang, 2013). Successive oxidation of 5-hmC by the TET enzymes produces 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC), which are found at a lower abundance in the genome (He et al., 2011). 

​​​Ultimately,  methylation, oxidation and repair provide a model for a cycle of dynamic cytosine modification, with mounting evidence for its significance in the biological processes known to involve active demethylation (Tan & Shi, 2012). 

​5-hmC analysis 

The most common approaches for studying DNA methylation are based on sodium bisulfite conversion. Sodium bisulfite conversion deaminates cytosine into uracil, but does not affect 5-mC. When the bisulfite-treated DNA is subjected to PCR, the uracil pairs to an adenine and is then amplified as a thymine, whereas the methylated cytosines remain unchanged. 

Downstream techniques (e.g. next generation sequencing and microarray) can be used to analyze the bisulfite converted DNA, where cytosines that are read as cytosine represent methylated cytosine, while those that are read as thymine represent unmethylated cytosine.

However, upon the popularization of 5-hmC, it was realized that conventional assays, including bisulfite and sequencing (BS-seq), were not able to distinguish between 5-mC and 5-hmC (Huang et al., 2010 and Jin et al., 2010). Hence, the distinction has become a new frontier in the understanding of the regulation of gene expression. 

Two novel, bisulfite-based technologies have recently emerged that address the challenge of mapping 5-hmC:

  • Oxidative bisulfite sequencing (oxBS-seq) can quantitatively distinguish 5-mC and 5-hmC marks at single-base resolution in genomic DNA
    (Booth et al., 2013). In oxBS-seq, specific oxidation of 5-hmC to 5-fC and conversion of the newly formed 5-fC to uracil (under bisulfite conditions) allows 5-hmC to be discriminated from 5-mC. A positive readout of actual 5-mC is gained from a single oxBS-seq run.
  • TET-assisted bisulfite sequencing (TAB-seq) involves β​-glucosyltransferase (β-GT)-mediated protection of 5-hmC (glucosylation) and recombinant TET mediated oxidation of 5-mC to 5-caC
    ​(Yu et al., 2012). After subsequent bisulfite treatment and PCR amplification, both cytosine and 5-caC (derived from 5-mC) are converted to thymine, whereas 5-hmC reads as cytosine.

Although these approaches address the core issue of distinguishing 5-mC from 5-hmC, the additional oxidative step combined with bisulfite can introduce another layer of potentially variability and sample loss.

Researchers are working towards both improving the above methods and developing new ones for 5-hmC detection. Some of the more recent advancements include:

  • A unique 5-hmC dependent restriction endonuclease (AbaSI) coupled with sequencing (Aba-seq), with the specificity of AbaSI enabling sensitive detection of 5-hmC at low-occupancy regions (Sun et al., 2013).
  • Significant development in antibodies specific to 5-hmC, which has led to the development and application of a technique called hydroxymethylated DNA immunoprecipitation (hMeDIP) ​(Nestor & Meehan, 2014). This technique parallels MeDIP, a popular technique to study 5-mC
    (Weber et al., 2005).
  • A genetically engineered protein pore (nanopore) from Mycobacterium smegmatis porin A (MspA) that can use ions to distinguish and map 5-mC and 5-hmC on single strands of DNA (Lazlo et al., 2013) with error rates suggesting 5–19 reads are needed for genome wide examination
    (Schreiber et al., 2013).​

Although these methods are very promising, they still have their limitations. For example, the nanopore technology is still in very early stages of development, while other techniques suffer from selection biases (AbaSI for certain CpGs and 5-hmC for antibody specificity).

Insight into 5-hmC biological mechanisms

Investigations into 5-hmC have begun to yield great insight into our understanding of development and complex disorders.

Recent investigation has generated single-base resolution 5-hmC maps that show that 5-hmC marks regulatory regions in the developing fetal brain genome (Lister et al., 2013). These regions then go on to be CpG demethylated by TET2 and activated in the adult brain.

Ultimately, this suggests that there are 5-hmC signatures for developmentally important regions (Tan & Shi, 2012). Furthermore, 5-hmC may have great implications for our understanding of disease, given its environmentally responsive nature (Blaschke et al., 2013), as evidenced by its dynamic response to cell media conditions and life experience.


  • Blaschke K, Ebata KT, Karimi MM, Zepeda-Martinez JA, Goyal P, Mahapatra S, Tam A, Laird DJ, Hirst M, Rao A, Lorincz MC and Ramalho-Santos M (2013). Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226.
  • Booth MJ, Ost TW, Beraldi D, Bell NM, Branco MR, Reik W, Balasubramanian S (2013). Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8, 1841–1851.
  • Gavin DP, Chase KA and Sharma RP (2013). Active DNA demethylation in post-mitotic neurons: a reason for optimism. Neuropharmacology 75, 233-245.
  • Globisch D, Munzel M, Muller M, Michalakis S, Wagner M, Koch S, Bruckl T, Biel M, Carell T (2010). Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367.
  • He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, Dai Q, Song CX, Zhang K, He C and Xu GL (2011). Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307.
  • Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR and Rao, A (2010). The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888.
  • Iurlaro M, Ficz g, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S and Reik W (2013). A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119.
Iurlaro M, Ficz g, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S and Reik W (2013). A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol 14, R119.
  • Jin SG, Kadam S, Pfeifer GP (2010). Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125.
  • Kohli RM and Zhang Y (2013). TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502, 472–479.
  • Kothari RM and Shankar V (1976). 5-methylcytosine content in the vertebrate deoxyribonucleic acids: species specificity. J. Mol. Evol. 7, 325–329.
  • Kriaucionis S and Heintz N (2009). The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930.
  • Laszlo AH, Derrington IM, Brinkerhoff H, Langford KW, Nova IC, Samson JM, Bartlett JJ, Pavlenok M, Gundlach JH (2013). Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl. Acad. Sci. U. S. A. 110, 18904–18909.
  • Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, Lucero J, Huang Y, Dwork AJ, Schultz MD, Yu M, Tonti-Filippini J, Heyn H, Hu S, Wu JC, Rao A, Esteller M, He C, Haghighi FG, Sejnowski TJ, Behrens MM and Ecker JR (2013). Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905.
  • Nestor CE and Meehan RR (2014). Hydroxymethylated DNA immunoprecipitation (hMeDIP). Methods Mol. Biol. 1094, 259–267.
  • Penn NW, Suwalski R, O'Riley C, Bojanowski K, Yura R (1972). The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid. Biochem. J. 126, 781–790.
  • Pfeifer GP, Kadam S, Jin SG (2013). 5-Hydroxymethylcytosine and its potential roles in development and cancer. Epigenet. Chromatin 6, 8935–8936.
  • Schreiber J, Wescoe ZL, Abu-Shumays R, Vivian JT, Baatar B, Karplus K, Akeson M (2013). Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands. Proc. Natl. Acad. Sci. U. S. A. 110, 18910–18915.
  • Sun Z, Terragni J, Borgaro JG, Liu Y, Yu L, Guan S, Wang H, Sun D, Cheng X, Zhu Z, Pradhan S and Zheng Y (2013). High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell. Rep. 3, 567–576.
  • Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L and Rao A (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935.
  • Tan L. and Shi YG (2012). Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139, 1895–1902.
  • Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL and Schubeler D. (2005). Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853–862.
  • Wyatt GR and Cohen SS (1952). A new pyrimidine base from bacteriophage nucleic acids. Nature 170, 1072–1073.
  • Yu M, Hon GC, Szulwach KE, Song CX, Jin P, Ren B and He C (2012). Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat. Protoc. 7, 2159–2170.
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