Figure 1. Irg1 expression reduces Tet2-catalyzed 5hmC production in cells.

a, Schematic diagram of experimental procedures to determine the effect of Irg1 expression on global histone and DNA de/methylation.

b, Overexpression of Irg1, but not catalytic-inactive mutant, results in elevated intracellular levels of ITA and succinate. Flag-tagged WT or H103A/K207E/K272E mutant Irg1 (referred to as Irg1^mut) was transiently overexpressed in HEK293T cells, and the intracellular levels of metabolites were determined by LC-MS/MS.

c, Overexpression of Irg1 does not significantly affect global histone demethylation markers. Transfected HEK293T cells in (b) were subjected to histone methylation quantification by MRM-based LC-MS/MS.

d, Overexpression of Irg1, but not Irg1^mut, inhibits Tet2 activity. Flag-tagged wild-type Irg1 or catalytic defect mutant was co-expressed with HA-mTET2^CD in HEK293T cells. The ectopically expressed proteins of Irg1 and Tet2 proteins were verified by western-blot using indicated antibodies.

e, f, Overexpression of Irg1, but not Irg1^mut, inhibits Tet2-catalyzed 5hmC production. Flag-tagged wild-type Irg1 or catalytic defect mutant was co-expressed with HA-mTET2^CD in HEK293T cells and was then subjected to detect 5hmC by immunofluorescence staining using indicated antibodies(e) Data shown represent 3 independent experiments’ in e. Scale bar, 50μM.Genomic DNA from the transfected cells in (d) were also subjected to 5hmC and 5mC quantification by LC-MS/MS (f).

Data shown in b, c, and f are average values with S.D. of n = three independent experiments. Asterisks denote statistical significance with one-way (f) or two-way ANOVA (b) for multiple comparisons. *denotes p < 0.05, ***denotes p < 0.001, ****denotes p < 0.0001 for the indicated comparison. n.s. = not significant.