5-Hydroxymethylcytosine is a predominantly stable DNA modification

Abstract

5-Hydroxymethylcytosine (hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid-chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.

Figures

Figure 1. Global levels of mC and hmC do not change during the cell cycle

(a) An overview of the LCMS method for quantification of modified cytosines. Genomic DNA, isolated from cells, is digested in a single step with a mixture of enzymes to generate 2′-deoxynucleosides, which are separated on an ultra high performance liquid chromatography (UHPLC) column and quantified using tandem mass spectrometry. The levels of mC and hmC are expressed as a percentage of total cytosines. For calibration curves see Supplementary Figure S1. (b) Global levels of mC and hmC present in DNA isolated from HCT116 and MCF7 cells arrested in G1/S interphase and allowed to progress through one cell cycle. The shaded background indicates in which phase the majority of cells was found by fluorescence activated cell sorting (FACS) analysis (Supplementary Fig. S2). Shown are mean ± SEM of 3 and 2 biological replicates for HCT116 and MCF7, respectively, and at least 2 technical replicates per sample. (c) Global mC and hmC levels in HCT116 and mES cells sorted by the DNA content into G1/0, early S, late S and G2/M phases. Shown are mean ± SEM of 2 biological and 2 technical replicates per sample. See also Supplementary Figure S4.

Figure 2. DNA methylation and mC oxidation activities occur with a marked time difference

(a) An overview of the labeling strategy for measuring the timing of mC oxidation. Cells were grown in a medium containing L-methionine-(methyl-13C,d3), which leads to incorporation of stable isotopes into newly formed mCs in genomic DNA via S-adenosylmethionine (SAM). Subsequent LCMS analysis using accurate masses of labeled and unlabeled species provides mC and hmC labeling ratios (% mC* and hmC*). This in turn allows following the formation of hmC on a newly methylated DNA. (b) An example of extracted ion chromatograms from a DNA digest from mES cells labeled with L-methionine-(methyl-13C,d3) showing all analytes (heavy and light mC and hmC), and their corresponding retention times and mass transitions. The coefficient of variation for 3 technical replicates is typically less than 2%. MS signal intensity (arbitrary units) is shown on the y axis. (c) HCT116, MCF7 and mES cells were grown in the heavy medium for more than 14 days. The gap between mC and hmC labeling curves reveals that there is a timing difference between mC formation and its oxidation to hmC. (d) Detailed analysis of the first 10 h of labeling shows that in cancer cell lines and mES cells, it takes at least 4 h and 2 h, respectively, to begin oxidizing newly methylated DNA. Shown are mean and S.E.M. from 2 biological replicates (HCT116 and mES) or 2 technical replicates (MCF7). See also Supplementary Figures S8 and S9 for the detection limits of our LCMS, and more biological replicates for HCT116 and MCF7 cells.

Figure 3. The majority of genomic hmC is stable

(a) Asynchronous HCT116 and MCF7 cells were labeled with L-methionine-(methyl-13C,d3) for 2 h and then grown in a light medium for several days. The bulk of hmC is derived from mC that is 15-25 h old, and persists in the genome until the labeling ratio gets diluted due to proliferation (i.e. generation of more unlabeled hmC). Dotted lines represent mC and hmC labeling in HCT116 cells that grew to confluency and stopped dividing, showing how stable the majority of hmC is in DNA. Shown are mean ± SEM of 2-3 technical replicates between 3-14 h of the experiment. The coefficient of variation between technical replicates is typically less than 2%. See also Supplementary Figure S10 for 6 and 10 h pulses in HCT116. (b) Labeling of undifferentiated and differentiating mES cells shows similar mC and hmC labeling profiles as for the differentiated cancer cells in (a). Shown are mean ± SEM of 2 technical replicates. Single replicate is shown for hmC in undifferentiated mES cells. (c) A breeding pair of wild-type animals was fed with a diet containing L-methionine-(methyl-13C,d3) for 117 days, and a range of tissues was analyzed by LCMS. The low labeling efficiency in slowly dividing and non-dividing tissues indicates that the majority of both mC and hmC must be stable in vivo. Gut-SI = small intestine. Shown are mean ± SEM of 2 technical replicates. See also Supplementary Figure S12. (d) mC and hmC labeling ratios in newborns (1 d old, parents labeled for 52 d before fertilization). Shown are mean ± SEM of 2 pups.

Figure 4. Isotopic labeling of DNA in vivo confirms the stability of hmC and reveals a relationship between global hmC levels and proliferation

(a) Linear correlation between proliferation rate (estimated from % mC*) and global levels of hmC in tissues from WT adult mice labeled for 117 d (male and female). Brain outlier (red) was omitted from the correlation coefficient. Shown are mean ± SEM of 2 animals on the y axis (except for prostate and testis), and a mean %mC* was used on the x axis. (b) Immunohistochemistry for hmC and the proliferation marker Ki67. Shown are details of the dentate gyrus in the hippocampus, hepatic triad, splenic germinal centre, crypts and villi in the small intestine, and the cortico-medullary junction in the thymus. hmC is abundant in the differentiated, non-proliferating cell types and low in proliferating Ki67 positive cells. Horizontal bars indicate 100 μm (1 mm in inset image). (c) A model explaining the reduction of global hmC levels in faster proliferating cells and tissues compared to slowly proliferating ones (e.g. tumour vs. healthy tissue) based on our findings about the timing of DNA oxidation and the persistence of hmC in the genome. According to this model, the average age of DNA in a population of differentiated cells will be the major determining factor of global hmC content.