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Abstract
Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer’s disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples.
Highlights
Since the discovery of cytosine demethylation, hydroxymethylcytosine has been identified as a possible “sixth base” due to its apparent involvement in gene expression [1], especially in neuronal tissue where hmC levels appear to be the highest [2]
Scientists have used high performance liquid chromatography (HPLC) to analyze global cytosine methylation, which overlooked three of the five cytosine variants involved in the demethylation cycle
Two reversed phase HPLC columns were tested in the optimization of a technique to separate the deoxynucleoside standards; an Agilent C18 (50 × 3 mm, 1.8 μm particle size) and a Phenomenex (Torrance, CA, USA) Luna Phenyl Hexyl (150 × 4.6 mm, 5 μm particle size)
Summary
Since the discovery of cytosine demethylation, hydroxymethylcytosine (hmC) has been identified as a possible “sixth base” due to its apparent involvement in gene expression [1], especially in neuronal tissue where hmC levels appear to be the highest [2]. These findings have prompted the need for methodologies that are able to distinguish between highly similar cytosine analogs. It has been determined that cytosine, following methylation, can undergo demethylation through the action of the ten-eleven translocation (TET) family of enzymes [9,10,11] In this process, mC is sequentially oxidized to hmC, formylcytosine (fC) and carboxycytosine (caC) (Figure 1). One must differentiate between the five variants of cytosine when determining methylation status
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