Quantitation and mapping of the epigenetic marker 5‐hydroxymethylcytosine

Abstract

We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.

5-Hydroxymethylcytosine (5hmC), or How to Identify Your Favorite Cell

“…Early methods such as immunofluorescence (IF) and mass spectrometry (MS) allowed researchers to assess global levels of 5hmC, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) is still the most accurate method for 5hmC global detection [10]. However, the development of locus-specific methods has played a pivotal role in our comprehension of the functional role of 5hmC.…”

Hydroxymethylation and tumors: can 5-hydroxymethylation be used as a marker for tumor diagnosis and treatment?

“…When 5mC in cfDNA was sequenced with bisulfite as a high background level inhibitory marker, the results were severely disturbed by DNA degradation [33]. However, unlike 5mC, 5hmC can be mapped with a low level of DNA and highly sensitive selective chemical markers, and the identified 5hmC has cancer type specificity [34][35][36]. Changes in 5hmC rich in enhancers, gene bodies, and promoters are closely related to changes in gene expression levels [37].…”

Analytical epigenetics: single-molecule optical detection of DNA and histone modifications

“…When the respective cofactor in this reaction, uridine diphosphoglucose (UDPG), is modified, new chemical functionalities can be introduced [22,23]. In particular, an azide-modified glucose offers a universal linkage point through click chemistry (Huisgen cycloaddition).…”

“…The nucleobase most-thoroughly studied by covalent epigenetic labeling is 5hmC, owing to the ability of T4 β-glucosyltransferase (βGT) to selectively place a glucose moiety onto the hydroxyl group of 5hmC. When the respective cofactor in this reaction, uridine diphosphoglucose (UDPG), is modified, new chemical functionalities can be introduced [22,23]. In particular, an azide-modified glucose offers a universal linkage point through click chemistry (Huisgen cycloaddition).…”