Every mammalian cell seems to contain 5-Hydroxymethylcytosine, but the levels vary significantly depending on the cell type. The highest levels are found in neuronal cells of thecentral nervous system.[5][6][7] The amount of hydroxymethylcytosine increases with age, as shown in mousehippocampus andcerebellum.[5][8]
The exact function of this nitrogen base is still not fully elucidated, but it is thought that it may regulategene expression or promptDNA demethylation. This hypothesis is supported by the fact that artificial DNA that contains 5-hydroxymethylcytosines (5hmC) can be converted into unmodified cytosines once introduced into mammalian cells.[9] Moreover, 5hmC is highly enriched inprimordial germ cells, where it apparently plays a role in global DNA demethylation.[10] Additionally, 5-Formylcytosine, an oxidation product of 5-Hydroxymethylcytosine and possible intermediate of an oxidative demethylation pathway was detected in DNA from embryonic stem cells,[11] although no significant amounts of these putative demethylation intermediates could be detected in mouse tissue.[7] 5-Hydroxymethylcytosine may be especially important in thecentral nervous system, as it is found in very high levels there.[7] Reduction in the 5-Hydroxymethylcytosine levels have been found associated with impaired self-renewal in embryonic stem cells.[12] 5-Hydroxymethylcytosine is also associated with labile, unstable nucleosomes which are frequently repositioned during cell differentiation.[13]
The accumulation of 5-hydroxymethylcytosine (5hmC) in post-mitoticneurons is associated with “functional demethylation” that facilitatestranscription andgene expression.[14] The term “demethylation,” as applied to neurons, ordinarily refers to the replacement of5-methylcytosine (5mC) bycytosine inDNA that can occur through a series of reactions involving aTET enzyme as well as enzymes of the DNAbase excision repair pathway (seeEpigenetics in learning and memory). “Demethylation” of 5mC in DNA most often results in the promotion of expression of genes with neuronal activities. “Functional demethylation” refers to the replacement of 5mC by 5hmC, ordinarily a single-step TET-mediated reaction, that also facilitates gene expression, an effect similar to that of “demethylation.”
Phages probably evolved to use 5hmC to avoid recognition by most restriction enzymes in bacteria. TheT4 phage uses 5hmC exclusively during replication, addingglycosylation to the hydroxyl group to further complicate the moiety.[15] Some bacteria have in turn evolvedrestriction enzymes specific for sites containing 5hmC. One prominent example is PvuRts1I, originally identified in 1994.[16]
5-Hydroxymethylcytosine was observed by Skirmantas Kriaucionis, an associate at the Heintz lab, who was looking for levels of5-methylcytosine in two different neuron types. He discovered a significant amount of an unknown substance instead, and after conducting several tests, identified it as being 5-hydroxymethylcytosine.[17]
The lab of L. Aravind used bioinformatic tools to predict that the Tet family of enzymes would likely oxidize 5-methylcytosine to 5-hydroxymethylcytosine.[18] This was demonstratedin vitro and in live human and mouse cells by scientists working in the labs of Anjana Rao andDavid R. Liu.
5-Hydroxymethylcytosine was originally observed inmammals in 1972 by R. Yura,[19] but this initial finding is dubious. Yura found 5-hmC present at extremely high levels in rat brain and liver, completely supplanting 5-methylcytosine. This contradicts all research conducted on mammalian DNA composition conducted before and since, including the Heintz and Rao papers, and another group was unable to reproduce Yura's result.[20]
With the discovery of 5-hydroxymethylcytosine some concerns have been raised regarding DNA methylation studies using the bisulfite sequencing technique.[21] 5-hydroxymethylcytosine has been shown to behave like its precursor, 5-methylcytosine, inbisulfite conversion experiments.[22] Therefore, bisulfite sequencing data may need to be revisited to verify whether the detected modified base is 5-methylcytosine or 5-hydroxymethylcytosine. In 2012 the lab ofChuan He discovered a method to solve the problems of 5-hydroxymethylcytosine being detected as 5-methylcytosine in normal bisulfite conversion experiments using the oxidative properties of the Tet-family of enzymes, this method has been termedTAB-seq.[23][24]
In June 2020, Oxford Nanopore added a hydroxymethyl cytosine detection model to their research basecaller, rerio, allowing old signal-level data from any R9+ nanopore runs to be re-called to identify 5hmC.[25]
As of 2024, methods to characterize the presence of 5-hmC include:[26]
Enrichment:immunoprecipitation (hMeDIP, CMS-DIP); affinity pulldown (GLIB, hmC-Seal), oligotagging with near-base resolution (Jump-seq, TOP-seq); hmC-CATCH with base-resolution
^abMünzel M, et al. (July 2010). "Quantification of the Sixth DNA Base Hydroxymethylcytosine in the Brain".Angew. Chem. Int. Ed.49 (31):5375–5377.doi:10.1002/anie.201002033.PMID20583021.
^Pfaffeneder T, Hackner B, Truss M, Münzel M, Müller M, Deiml CA, Hagemeier C, Carell T (30 June 2011). "The Discovery of 5-Formylcytosine in Embryonic Stem Cell DNA".Angew. Chem. Int. Ed.50 (31):7008–7012.doi:10.1002/anie.201103899.PMID21721093.
^Jin SG et al. (Jun 2010) "Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine." Nucleic Acids Res. 2010 Jun 1;38(11):e125
^Erlitzki N, Kohli RM (2024). "An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic DNA".Epigenome Editing. Vol. 2842. pp. 325–352.doi:10.1007/978-1-0716-4051-7_17.
