Fig 2: IDH2 mediates D2HGDH effects on histone and DNA methylation and HIF1α hydroxylation.(a) Western blot analysis of IDH1 and IDH2 in subcellular fractions of D2HGDH models (knockdown, left panel; ectopic expression, right panel) shows modulation of IDH2 levels. Densitometric quantification is shown at the bottom. (b) Top—western blot analysis of IDH2 KD cells expressing an empty-MSCV vector or WT D2HGDH. Middle—Expression of WT D2HGDH decreased H3K methylation (compare lanes marked with a red star); IDH2 KD restored the H3K methylation levels in these cells. Bottom—expression of WT D2HGDH increased HIF1α hydroxylation and decreased total HIF1α levels (compare lanes marked with a red star); IDH2 KD reversed the increase in HIF1α hydroxylation (and decrease in total HIF1α) associated with expression of D2HGDH WT. (c) Cells expressing D2HGDH-WT (and an empty pLKO.1) displayed a significantly higher abundance of 5hmC marks (top) and a concomitant decrease in global DNA methylation (botton) than MSCV-pLKO.1 controls (marked by red star; P<0.001 two-tailed, Student's t-test). IDH2 KD significantly lowered or increased the levels of 5hmC and 5mC marks, respectively, in D2HGDH-WT cells (P<0.0001, ANOVA, P<0.001 Bonferroni's multiple comparison test), with a more modest change in MSCV-expressing controls. These data are mean and s.d. of an assay performed in triplicate, which was confirmed in an independent biological replicate. The data from the WBs were confirmed in two to three biological replicates. (d) Top—western blot analysis of IDH2 ectopic expression in D2HGDH KD cells. Middle—D2HGDH KD elevated H3K methylation (compare the three first lanes) and this change was abrogated by expression of IDH2. Bottom—D2HGDH KD decreased HIF1α hydroxylation and increased its total levels (compare the three first lanes); these changes were absent in D2HGDH KD cells ectopically expressing IDH2. (e) D2HGDH KD decreased the abundance of 5hmC marks (top) and increased global DNA methylation 5mC (bottom; <0.0001, ANOVA); expression of IDH2 restored these values to that of control (pSIL-MSCV) isogenic cells. These data are mean and s.d. of an assay performed in triplicate, confirmed in a biological replicate. The western blot data shown in d were confirmed in biological replicates.

Fig 3: IDH1 binds to GA/AU-rich single-stranded RNA. (A) GA- and AU-rich RNA motifs are enriched in IDH1’s RNA targets in all sequences or in the 3' UTR regions, respectively. (B–E) EMSA analysis of direct binding of purified wild-type to various fluorescence-labeled RNA/DNA probes as indicated. (B) EMSA with the 5 × GAA ssRNA probe containing the overall motif #1. (C) EMSA with ssRNA probes with 2–5 copies of GAA(A) nucleotides. (D) Binding isotherms for IDH1-RNA interactions. Each data point is shown as mean ± SD (n = 3 biological replicates). Error bars represent standard deviations (SD), some of which are too small to be visualized. (E) Summary of RNA-binding activities of IDH1 toward various ssRNA probes used above. Dissociation constants (Kd) are shown. N.D., not detected. GAA and AUUU units are underlined. (F) Relative m6A level of IDH1 RNA targets in ESCs was determined by HPLC-MS/MS following FBioRIP assays. Data are shown as mean ± SD, n = 3. (G) Comparison of RNA-binding activities of wild-type (WT) and oncogenic R132H mutant of IDH1 to the 5× GAA ssRNA probe as shown in the panel (B).

Fig 4: Wild type and mutant IDH1 and 2 homo- and heterodimers show differences in their ability to generate NADPHHEK293FT cells were transfected with the indicated IDH1 (a) or IDH2 (b) BiFC constructs or left untransfected (endogenous IDH control: gray lines) and assessed for their ability to generate NADPH in the presence of excess NADP+ and isocitrate using an Isocitrate Dehydrogenase Activity Colorimetric Assay Kit as per the manufacturer’s protocol. 0.25 µg of each DNA construct was transfected in a 24-well plate format (0.5 µg total). For 50% DNA samples, 0.25 µg of each single DNA construct was transfected. (c) 0.25 µg of each of VN173-IDH1WT and VC155-IDH1R132H were co-transfected into HEK293FT cells along with the indicated increasing concentration of IDH1WT-HA. Cells were assessed for their ability to generate NADPH using an Isocitrate Dehydrogenase Activity Colorimetric Assay Kit as per the manufacturer’s protocol. (d) 0.25 µg of each of IDH2WT-VN173 and IDH2R172K-VC155 were co-transfected into HEK293FT cells along with the indicated increasing concentration of IDH2WT-HA. Cells were assessed for their ability to generate NADPH using an Isocitrate Dehydrogenase Activity Colorimetric Assay Kit as per the manufacturer’s protocol. Data is representative of at least 3 independent experiments.

Fig 5: SplitLuc CETSA assay development. (A) Schematic overview of the ultra-high-throughput cellular thermal shift assay using split NanoLuc. A peptide (86, red text) previously reported as having strong affinity for a large fragment of NanoLuc (11S) was modified to have Gly-Ser linkers on each side (86b-tag). (B) 2-HG production in cells expressing tagged IDH1(R132H) or controls (EGFP or no DNA). 86b was fused to either the N- or C-terminus of IDH1(R132H). As a control for the 86b tag a FLAG tag was fused to the C-terminus of IDH1(R132H). Enzymatic activity of the tagged transgene was assessed by measuring 2-HG in the medium 72 h post-transfection (n = 3 per variant, x-axis). Transgene expression levels were measured by luminescence resulting from NanoLuc complementation (y-axis). (C) Thermal stability of endogenous (wild-type) and transfected (R132H mutant) IDH1 by immunoblot using pan (top) or R132H specific (middle, N-tag; lower, C-tag) antibodies. Untransfected/unheated HEK293T cells (endogenous WT IDH1) serve as the control (CTRL) sample. (D) Densitometric analysis of immunoblot in (C) enables quantification of Tagg for endogenous and 86b-tagged IDH1(R132H). (E) Thermal profiles of 86b-IDH1(R132H) and IDH1(R132H)-86b extracted via freeze-thaw or NP40-mediated lysis. (F) Aggregated IDH1(R132H) is complementation incompetent and does not require removal by centrifugation. After heating to 65 °C for 3.5 min, samples were centrifuged at various speeds for 20 min and complementation was assessed. (G) Treatment with 1 µM AG-120, an inhibitor of mutant IDH1, stabilizes N- and C-tagged IDH1(R132H). Cells were treated for 1 h and heated for 3.5 min (mean ± SD, n = 3).