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 embryonic stem (ES) cells has necessitated a reevaluation 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 on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.

The effect of morphine upon DNA methylation in ten regions of the rat brain

“…35 While 5-hmc is an intermediate in active DNA demethylation, 5-mc and 5-hmc levels are not inherently correlated 36 and therefore our observations of decreased 5-mc and 5-hmc are not contradictory. Furthermore, our results could also be the product of the direct role of 5-hmc in RNA splicing 37 and gene expression through inhibition of chromatin remodelling 38 , which may offer an alternative explanation for our observations.…”

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

“…5hmC enrichment within the H19 gene body was also analyzed since previous studies have identified the region as enriched for 5hmC. 30 As the ICR1 is known to be enriched for 5mC, the H19 gene body is enriched for 5hmC and the GAPDH promoter is depleted for both modifications; 30 enrichment at these regions acted as positive and negative controls for each cytosine modification. Primer details are given in Table 1.…”

“…28 5-hydroxymethylcytosine (5hmC) is a recently described cytosine modification that may be an intermediate in the DNA demethylation pathway 29 and is present in the placenta. 30 Since commonly used methods of assessing DNA methylation using bisulfite conversion do not discriminate between 5mC and 5hmC, 31 previous studies reporting altered DNA methylation at placental DMRs in association with fetal growth may be confounded. We used specific enrichment techniques to map 5mC and 5hmC at the DMRs controlling the expression of IGF2 and CDKN1C in human placenta, and additionally explored relationships between 5mC, 5hmC, gene expression, and fetal growth across the full range of normal birth weight.…”

Analysis of DNA modifications in aging research

“…f CG methylation across promoters often varies greatly across a relatively narrow region with lowest methylation typically observed around the transcription start site (TSS), even in un-expressed genes CH sites and hydroxymethylation of CG sites are prevalent in several organ systems, particularly the central nervous system (Lister et al 2013;Kinde et al 2015). The high levels of hmCG and mCH in the brain stand in contrast to other organs (Nestor et al 2012) and suggest the possibility that the epigenomics of the aging brain is substantially different from other tissues (Masser et al 2017a). CG dinucleotides occur far less frequently (< 1% of dinucleotide pairs) than any other dinucleotide pairs (Bird 1980;Karlin and Mrázek 1997) and are clustered together in regions termed CG islands (Lander et al 2001).…”