5-Hydroxymethylcytosine: a new kid on the epigenetic block?

Abstract

The discovery of the Ten-Eleven-Translocation (TET) oxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG-island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI-HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta-analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a 'true' epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.

Figure 1

Heatmap representation of TET1 enrichment in CGI promoters (top panel) and bivalent promoters (middle panel). Heatmap representation of TET1 DnaseI-HS (ENCODE-ZhBTc4 cell line), hmC, and mC enrichment in TET1-N-binding sites (bottom panel). All the heatmaps are from +5 to −5 kb flanking TSSs of CGI, bivalent promoters or TET1-N centered binding sites, reads were summed in 100 bp sliding windows. Datasets used in the analysis are indicated.

Figure 2

Heatmap representation of 5hmC and 5mC enrichment at CGI promoters and bivalent promoters depicted as in Figure 1.

Figure 3

Hierarchical clustering of pairwise R² correlation on 5hmC and 5mC genome-wide profiling studies in mouse ESCs. (A) Hierarchical clustering and heatmap representation in the full genome (1 kb sliding windows) (B) CGI promoters (±500 bp sliding window from TSS).

Figure 4

5hmC and 5mC distributions at CA simple repeats and bivalent promoters. (A) Genome browser view of a CA simple repeat element with read distributions for 5hmC and 5mC using datasets as indicated. (B) 5hmC and 5mC distributions at CA simple repeats and in (C) bivalent promoters. All the distribution plots are from +5 to −5 kb flanking CA simple repeats centered (B) or TSSs bivalent promoters (C), reads were summed in 100 bp sliding windows.

Figure 5

Methylation score distribution in CA-repeats from bisulfite-seq studies in human ESCs. Datasets used in the analysis are indicated. Only cytosines with a coverage higher than 1 are included.