Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry

Abstract

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3-8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast.

Figure 1

Electron impact (EI) full-scan mass spectra of TMS derivatives of cytosine (A) and 5mC (B). (C) Selective ion monitoring (SIM) mass spectrum of 5mC from genomic DNA of yeast strain of Y. lipolytica (CGMCC 2.1718). The fragment ions of m/z 269, 255, and 254 were used for monitoring 5mC.

Figure 2

Selective ion monitoring (SIM) chromatograms of nucleobases. (A) Chromatogram of 6 standard nucleobases. (B) Representative chromatogram for nucleobase mixture from genomic DNA of Y. lipolytica (CGMCC 2.1718). (C) Representative chromatogram for nucleobase mixture from genomic DNA of S. cerevisiae W1588-4C. U, uracil; T, thymine; C, cytosine; 5mC, 5-methylcytosine; A, adenine; G, guanine.

Figure 3

Selective ion monitoring (SIM) chromatogram of the TMS derivatives of individual nucleobase standards after acid hydrolysis. U, uracil; T, thymine; C, cytosine; 5mC, 5-methylcytosine; A, adenine; G, guanine.

Figure 4

(A) Optimization of derivatization temperature. (B) Optimization of derivatization time.

Figure 5

Genome-wide DNA methylation levels in Y. lipolytica (CGMCC 2.1718) and S. cerevisiae W1588-4C at different growth stages. The DNA methylation level was shown as average molar ratio of 5mC/(5mC + cytosine) ± SD (n = 3); p values were derived from Tukey’s test.