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. 2018 Oct 24;9:4429. doi: 10.1038/s41467-018-06841-7

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© The Author(s) 2018

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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Fig. 4 — DOT1L mediates α-HB-induced H3K79 hypermethylation. a α-HB upregulates H3K79 trimethylation. H3K79 methylation levels were analyzed in HaCaT and HT-3 cells upon 1 mM four LDHA-related metabolite treatments for 24 h. Ctrl, control; α-HB, sodium α-hydroxybutyrate; α-KB, sodium α-ketobutyrate; Lac, sodium l-lactate; Pyr, sodium pyruvate. H3K79 methylation / H3 ratio was quantified. b ROS increase H3K79 trimethylation. H3K79 methylation levels were analyzed in HaCaT and HT-3 cells upon 10 μM H₂O₂ for 6 h with or without 1 mM NAC pretreatment for 1 h as indicated. H3K79 methylation/H3 ratio was quantified. c E7 expression enhances H3K79 trimethylation. H3K79 methylation levels were analyzed in PHKs and HaCaT cells stably expressing vector or Flag-tagged HPV16/18 E7 coupled with or without 1 mM NAC treatment for 6 h. H3K79 methylation/H3 ratio was quantified. d DOT1L mediates nuclear LDHA-induced H3K79 hypermethylation. HeLa stable cells with LDHA knockdown and Flag-tagged Vec/WT/NLS/NES rescue were treated with or without 3 μM EPZ004777 for 48 h, combined with or without 1 mM sodium α-HB for 48 h as indicated. β-actin and histone H3 used as loading control in all immunoblots. e E7 triggers the binding of DOT1L with LDHA. Endogenous coimmunoprecipitation of DOT1L in HT-3 cells stably expressing vector or HPV16 E7 coupled with or without 1 mM NAC treatment for 6 h, and blotting of LDHA and DOT1L. f ROS trigger the binding of DOT1L with LDHA. Endogenous coimmunoprecipitation of DOT1L in HT-3 cells with or without 10 μM H₂O₂ for 6 h or 1 mM NAC for 6 h as indicated, followed by blotting with LDHA and DOT1L. g α-HB induces the binding of DOT1L with LDHA. Reversible endogenous coimmunoprecipitation of DOT1L and LDHA in HaCaT and HT-3 cells with or without 1 mM α-HB for 24 h, followed by blotting with LDHA and DOT1L. Results are representative of three independent experiments. All data are shown as mean ± SEM. The p values were determined by two-tailed t-test. The values of p < 0.05 were considered statistically significant. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. NS means non significant