Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA

Abstract

BackgroundThe discovery that 5-methylcytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) proteins has prompted wide interest in the potential role of 5hmC in reshaping the mammalian DNA methylation landscape. The gold-standard bisulphite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genome-wide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC. In this study, we provide a comparative analysis on brain DNA using three 5hmC genome-wide approaches, namely whole-genome bisulphite/oxidative bisulphite sequencing (WG Bis/OxBis-seq), Infinium HumanMethylation450 BeadChip arrays coupled with oxidative bisulphite (HM450K Bis/OxBis) and antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq). We also perform loci-specific TET-assisted bisulphite sequencing (TAB-seq) for validation of candidate regions.ResultsWe show that whole-genome single-base resolution approaches are advantaged in providing precise 5hmC values but require high sequencing depth to accurately measure 5hmC, as this modification is commonly in low abundance in mammalian cells. HM450K arrays coupled with oxidative bisulphite provide a cost-effective representation of 5hmC distribution, at CpG sites with 5hmC levels >~10%. However, 5hmC analysis is restricted to the genomic location of the probes, which is an important consideration as 5hmC modification is commonly enriched at enhancer elements. Finally, we show that the widely used hMeDIP-seq method provides an efficient genome-wide profile of 5hmC and shows high correlation with WG Bis/OxBis-seq 5hmC distribution in brain DNA. However, in cell line DNA with low levels of 5hmC, hMeDIP-seq-enriched regions are not detected by WG Bis/OxBis or HM450K, either suggesting misinterpretation of 5hmC calls by hMeDIP or lack of sensitivity of the latter methods.ConclusionsWe highlight both the advantages and caveats of three commonly used genome-wide 5hmC profiling technologies and show that interpretation of 5hmC data can be significantly influenced by the sensitivity of methods used, especially as the levels of 5hmC are low and vary in different cell types and different genomic locations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0123-7) contains supplementary material, which is available to authorized users.

Selective inhibition of CTCF binding by iAs directs TET-mediated reprogramming of 5-hydroxymethylation patterns in iAs-transformed cells

“…4D). These results are in line with other studies showing a positive correlation of hydroxymethylation levels with levels of gene expression (Colquitt et al, 2013; Tsai et al, 2014; Skvortsova et al, 2017). Indeed, other genes that had differential promoter hydroxymethylation from RRHP (though we were not able to amplify products from 5hme-DIP) also showed correlating changes in gene expression levels.…”

“…Not surprisingly, genes with low promoter 5hmC enrichment are least expressed, while genes with high 5hmC at the promoter are highly expressed. This observation is in agreement with earlier reports of positive correlation of 5hmC levels with promoter gene expression (Colquitt et al, 2013; Tsai et al, 2014; Skvortsova et al, 2017). We also observed low 5hmC levels within high CpG regions (only 16.25% of our dataset), which is in line with previous studies (Booth et al, 2012; Putiri et al, 2014).…”

Epigenetics applied to psychiatry: Clinical opportunities and future challenges

“…114,115 It is noteworthy that conventional BSbased arrays do not cover sufficient non-CpG nor allow distinguishing 'true' methylation from hydroxymethylation, thereby leading to mixed signals of both modifications. In that context, emerging technologies, such as oxidative BS 125 or Tet-assisted BS 126 along with the generalization of sequencing methodologies will likely provide new tools for investigation of these regulatory brain-specific marks.…”

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro hepatocyte differentiation

“…Comparisons with an FDR-adjusted P value below 0.05 were considered statistically significant. In addition, only DhMPs with at least 10% change in 5hmC were considered as significant for any given comparison, as this has been suggested as a threshold for the sensitivity of this technique [Skvortsova et al, 2017]. DEGs, DMPs, and DMRs were further analyzed to determine functional pathways and ontology enrichment using EnrichR [Chen et al, 2013].…”