The pattern of methylated cytosine residues in DNA provides an inheritable epigenetic code that regulates gene expression during development. The covalent addition of a methyl group at the 5-position of cytosine primarily occurs in the CpG dinucleotide, and is catalyzed by a family of DNA methyltransferases (Dnmts) including maintenance Dnmt1 and de novo
Dnmt3a and Dnmt3b. DNA methylation is involved in various biological processes such genomic imprinting, silencing of retroviral transposons, X chromosome inactivation, and cellular differentiation. Mechanistically, promoter methylation can lead to transcriptional repression directly by inhibiting transcriptional binding, or indirectly by recruiting various proteins including methyl CpG binding proteins (MBDs), co-repressors and histone modification enzymes involved in chromatin remodeling [3
]. Importantly, many studies have shown that DNA methylation is a dynamic process in cellular proliferation and differentiation, and is tightly regulated in normal development. Aberrant DNA methylation patterns and mechanisms are deleterious to the developing central nervous system (CNS) [8
Recently there has been renewed interest in another, related, mammalian DNA modification, 5-hydroxymethyl-cytosine (5hmC). Significant levels of 5hmC are found in the developed murine central nervous system and in embryonic stem cells [1
]. In vivo
addition of a hydroxyl group onto 5-methyl-cytosine (5mC) is catalyzed by 2-oxoglutarate oxygenase Tet1, Tet2, and Tet3 [1
]. There are also reports that 5hmC can be formed by other mechanisms beside Tet pathway, including UV irradiation of 5mC in aerated aqueous solution [15
] and DNA methyltransferase reaction of cytosine with formaldehyde [16
]. To date, only the Tet pathway has been demonstrated to produce 5hmC in mammalian genomic DNA.
Speculation that 5hmC is involved in the DNA demethylation pathway comes from the two reported mechanisms of converting 5hmC into C. Bacterial DNA methyltranferases catalyze the removal of formaldehyde from 5hmC, thus converting 5hmC to C [16
]. Another deformylation mechanism involves the photochemical hydration of 5hmC in basic solution [15
]. However, these two possible DNA demethylation mechanisms have yet to be confirmed in mammalian models.
Some of the most commonly used methods for profiling and quantification of DNA methylation, such as bisulfite sequencing and methylation-sensitive enzyme-based assays, are unable to distinguish between 5hmC and 5mC [1
]. Several methods have been used to measure the 5hmC levels in the genome: these include end-labeling followed by thin layer chromatography [1
], high performance liquid chromatography (HPLC) with UV detection [16
], enzymatic radioactive glycosylation labeling [2
], and single molecule, real-time sequencing [18
]. The thin layer chromatography method has the advantage of being low cost and simple, but requires the availability of radioactive substrates and the accuracy is not comparable to other available methods. The specificity of UV detection relies heavily on the chromatographic separation to avoid co-elution of other components, including other DNA and RNA nucleotides that may be present in biological samples. The glycosylation method is based on enzymatic incorporation of radio-labeled glucose into genomic 5hmC, with quantification by radioactive counting. However a complete enzymatic reaction cannot be readily assured and 5mC levels cannot be measured simultaneously. Independent measurement of 5hmC is possible with next generation sequencing, but the technology has yet to be perfected for accurate quantitation of many low abundant nucleotides including 5hmC.
Previous work demonstrated the precision, selectivity and sensitivity of liquid chromatography tandem mass spectrometry for measuring 5mC in biological samples, and as a diagnostic tool for cancer [19
]. Using this technique all known DNA (excluding 5hmC) and RNA components, have been separated, distinguished and independently quantitated [24
]. This approach allows DNA methylation to be measured both at the global [20
] and gene promoter regions [22
]. However, none of the previous reports include 5hmC. We were prompted to develop a fast, sensitive and accurate method to measure both 5mC and 5hmC levels to support ongoing work on epigenetic control of stem cells and neural development. Here we report the use of liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC-ESI-MS/MS-MRM) for the determination of genomic DNA methylation and hydroxymethylation. Separation of the deoxyribonucleosides is achieved within 6 minute using sub-two micron particle size reverse phase chromatography columns. In addition, mass-based detection discriminates between the three nucleoside bases of interest- 5hmC, 5mC and cytosine (C). The combination of LC and MS minimizes any possible cross-talk between the measurements of low abundant molecules (5hmC and 5mC) in the face of a chemically similar abundant species (C). Together, our data indicate that the MRM method provides unambiguous and independent quantification of 5hmC, 5mC, and C with high reproducibility and low limits of detection of around 0.5 fmol per sample. This limit of detection can be equated to 50 ng of digested genomic DNA to measure 5hmC levels at the 0.1% level. Furthermore, the method is relatively fast, requiring less than 48 hours from extracting genomic DNA (few hours to a day), to digesting genomic DNA into nucleoside components (1–2 hours), and measuring the 5hmC and 5mC levels using the MRM method (6 minutes per sample).