Technologies and applications for the assessment of 5-hydroxymethylcytosine

Jurkowski, Tomasz P. ORCID: https://orcid.org/0000-0002-2012-0240 and Friis, Ethan 2025. Technologies and applications for the assessment of 5-hydroxymethylcytosine. Tollefsbol, Trygve O., ed. Epigenetics Methods (Second Edition), Translational Epigenetics, Elsevier, pp. 239-257. (10.1016/B978-0-443-26759-8.00012-6)

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Abstract

DNA methylation is a crucial epigenetic modification involved in the control of genetic information and has been studied extensively over the past few decades. The balance between generation and removal of DNA methylation patterns is essential for human health. The discovery of TET enzymes has changed the view on DNA methylation as a very stable modification, as it showed that active DNA demethylation could occur through stepwise oxidation of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5 fC), and finally to 5-carboxylcytosine (5caC). This is then followed by the removal of the oxidized bases by thymine DNA glycosylase (TDG) through a base excision repair mechanism. Importantly, 5-hydroxycytosine and the further TET-oxidized bases are not only regarded as intermediates in the active DNA demethylation process but also recognized as separate epigenetic marks that carry epigenetic information distinct from 5-methylcytosine. The new modified cytosine bases, in particular, 5-hydroxymethylcytosine, have attracted a lot of attention in the scientific community, and numerous methods to map and quantify them have been developed and applied. They encompass methods to quantify 5hmC levels global in the bulk DNA sample, providing locus or base resolution for 5hmC mapping and quantification. In this chapter, I review current technologies used to quantify and map 5hmC, covering both global and focused 5hmC detection methods, that are based on different principles, including classical physicochemical analysis, protein domain or antibody-based affinity enrichment, enzymatic or chemical modification of 5hmC or restriction enzyme-based techniques. I further compare their specific advantages and disadvantages and advise which methodologies to use for specific experimental questions.

Item Type:	Book Section
Date Type:	Publication
Status:	Published
Schools:	Schools > Biosciences
Publisher:	Elsevier
ISBN:	9780443267598
Last Modified:	15 Oct 2025 09:31
URI:	https://orca.cardiff.ac.uk/id/eprint/181663

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