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. 2020 May 8;10:7822. doi: 10.1038/s41598-020-64700-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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5hmC genome-wide enrichment during hepatocyte differentiation. (A) Immunofluorescence of 5hmC in proliferative (Prolif), differentiating (Diff) and differentiating + IFC-305 (IFC) HepaRG cells. Quantification barplots of 5hmC signal are based on mean ± SEM from 3 fields per group. *Statistical difference (p < 0.05) compared with proliferative, or *⁺(p < 0.05) compared with proliferative and differentiating cells. (B) The same conditions were assessed for base-resolution 5mC/5hmC, using BS and OxBS followed by EPIC beadarray hybridization (see Methods). Proportion of signal loss after oxidation (mean oxBS signal / mean BS signal) was used as an estimation of global 5hmC in each condition. BS represents the variation between two BS technical replicates. Global distribution of 5hmC for one representative sample of each condition, according to CpG islands (CGI) (C) and transcription start sites (TSS) (D). In both cases, 5hmC levels are averaged across all hg19-annotated genomic regions. (E) Heatmap showing hydroxymethylome comparison between differentiating and proliferative cells. Differentially hydroxymethylated positions (DhMPs) were filtered by the magnitude of change in methylation (delta beta) of at least 20% and p-adjusted value <0.05. Two independent cultures were used for proliferative and differentiating cells, and three independent cultures for differentiating + 1 mM IFC-305.