DNA methylation in mammals occurs at the 5-position of the nitrogen base, cytosine, typically at CpG dinucleotide islands, to produce 5-methylcytosine. DNA methylation is a stable epigenetic mark that has important roles in mammalian development, differentiation and maintenance of cellular identity through the control of gene expression. When occurring in promoter regions, this modification provides stable gene silencing and reduces gene expression and transcription. DNA methylation is enzymatically catalyzed by a class of enzymes called DNA methyltransferases (DNMTs) utilizing S-adenosylmethionine (SAM) as the methyl group donor. SAM is then converted to S-adenosylhomocysteine (SAH) (Figure 1A). Changes in DNA methylation patterns are associated with some pathological conditions such as cancer, of which many forms are characterized by genome-wide hypomethylation and site-specific promoter hypermethylation.

DNA Methylation

Figure 1. A) Overview of the DNA methylation mechanism.

LC-MSMS chromatogram

Figure 1. B) LC-MS/MS chromatogram of 1 µg of A549’s DNA hydrolyzed by DNA Degradase Plus. The chromatogram shows selected base peaks of 5mdC at m/z 126.1, dC at m/z 112.1, and 2′-dG at m/z 152.1. The DNA hydrolysate was separated on an Agilent PoroShell 120 EC-C18 column with an isocratic solvent system composed of 90% H2O (0.1% (v/v) acetic acid) and 10% ACN (0.1% (v/v) acetic acid).

At Visikol, we developed a robust bioanalytical methodology based on LC-MS/MS for the rapid and accurate quantification of global DNA methylation in each sample. In brief, DNA is extracted from cancer cell lines or tissues and enzymatically hydrolyzed into its corresponding nucleosides by DNA Degradase Plus (Zymo Research, E2021). Nucleosides of interest, namely 2’-deoxycytidine (2’-dC), 5-methyl-2’-deoxycytidine (5mdC), and 2’-deoxyguanosine (2’-dG) are separated on an Agilent PoroShell 120 EC-C18 and quantified by a SCIEX QTRAP 4000 mass spectrometer in the multiple reaction monitoring (MRM) mode (Figure 1B). Linear dsDNA standards at a length of 897 bp (Zymo Research, D5405) were used to construct a calibration curve for global DNA methylation quantification. All DNA standards have the same sequence with either 100% dC, 100% 5mdC, or 100% 5hmdC. The calibration curve was constructed by mixing an increasing amount of hydrolyzed 5mdC in the presence of the same amount of hydrolyzed 2’-dC. To show the applicability of our methodology, two cancer cell lines, A549 (human lung carcinoma) and HCT116 (human colorectal carcinoma), were treated with 5 µM of the DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), for 72 hours, and their global DNA methylation status was assessed by our method.

As expected, 5-AZA treatment showed a 40% and 19% reduction in global DNA methylation status of A549 and HCT116, respectively, compared to their baseline methylation levels (Figure 1C).

If you are working on an epigenetic drug discovery project and need help quantifying your global DNA methylation, please reach out to our team to explore your epigenetic needs.

Figure 1. C) Effect of 5-AZA treatment (5 µM) on the global DNA methylation levels in two cancer cell lines, A549 and HCT116 after 72 hours of treatment.


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