Tissue-type is a major modifier of the 5-hydroxymethylcytosine content of human genes

Abstract

The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human ES cells has necessitated a re-evaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue-specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues we find that 5hmC patterns are tissue-specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue-type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (> 20-fold) dependent upon tissue-type. Our findings highlight tissue-type as a major modifier of 5hmC levels in expressed genes and emphasizes the importance of employing quantitative analyses in the study of 5hmC levels.