Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes

Abstract

The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe(2+) and 2-oxoglutarate-binding residues increased the Km values for these factors 30-80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400-500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.

Keywords: 5-hydroxymethylcytosine (5-hmC); AML; TET; epigenetics; fumarate; hypoxia; hypoxia-inducible factor (HIF); leukemia; succinate.

FIGURE 1.

Expression, affinity purification, and kinetic analyses of Tets. A–C, SDS-PAGE and Coomassie Blue analysis of the expression and affinity purification of recombinant Tet1 (A), Tet2 (B), and Tet3 (C). Cell lysate (lane 1), unbound proteins (lane 2), and FLAG-affinity purified proteins (lane 3) are shown. D and E, Michaelis-Menten curves and Lineweaver-Burk plots (inset) of Tet1 and Tet2 for oxygen.

FIGURE 2.

Kinetic analysis of AML-associated Tet2 mutants. A, SDS-PAGE analysis of purified recombinant Tet2 mutants H1302Y, D1304A, H1802R, R1817M, and R1817S. B and C, Michaelis-Menten curves and a Lineweaver-Burk plot (inset) of Tet2 mutants H1302Y and R1817M for Fe²⁺ and 2-oxoglutarate, respectively.

FIGURE 3.

Tets are susceptible to competitive inhibition by fumarate and succinate, resulting in lower global 5-hmC levels in cells treated with cell-permeable forms of these compounds. A and B, IC₅₀ curves of Tet1 and Tet2 for fumarate and succinate, respectively. C and D, HPLC-MS/MS quantitation of global 5-hmC (C) and 5-mC (D) levels in SK-N-BE(2) cells incubated with increasing concentrations of diethyl fumarate or dimethyl succinate for 48 h (n ≥ 3). 5-hmC and 5-mC quantitation graphs represent mean ± S.E. *, p < 0.05.

FIGURE 4.

Fumarate and succinate stabilize HIFαs and alter TET and HIF target gene expression. A and B, HIF-1α (A) and HIF-2α (B) protein levels determined by immunoblotting in SK-N-BE(2) cells exposed to hypoxia (1% O₂) or incubated with increasing concentrations of DEF or 5 mm DMS for 48 h. β-Actin was used as a loading control. C, IDH1 protein levels were determined by immunoblotting in SK-N-BE(2) cells incubated with increasing concentrations of DEF or DMS for 48 h. β-Actin was used as a loading control. D–F, qPCR analysis of TET1–3 mRNA expression levels in cells treated with increasing concentrations of DEF (D), DMS (E), or exposed to hypoxia (1% O₂) (F) for 48 h (n ≥ 3). G-I, qPCR analysis of selected HIF target genes in cells exposed to hypoxia (1% O₂) (G) or treated with increasing concentrations of DEF (H) or DMS (I) for 48 h (n ≥ 3). All graphs represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 5.

TETs regulate HIF target gene expression. A and B, qPCR analysis of TET1 (A) and TET3 (B) mRNA expression levels in SK-N-BE(2) cells following siRNA knockdown of TET1 (A) or TET3 (B) (n = 3). C and D, HLPC-MS/MS quantitation of global 5-hmC (C) and 5-mC (D) levels in SK-N-BE(2) cells following siRNA knockdown of TET1 or 3 and DEF or DMS treatment (n = 3). E and F, qPCR analysis of VEGFA and HK2 mRNAs following knockdown of TET1 or TET3 and DEF or DMS treatment (n = 3). G, qPCR analysis of BNIP3, PGK1, and ENO1 mRNA expression in SK-N-BE(2) cells following siRNA knockdown of TET1 or TET3 (n = 3). All graphs represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 6.

Fumarate and succinate regulate HIF target gene expression via TETs. FH and SDH mutations with impaired enzyme activity are found in various cancers, where they cause the accumulation of fumarate or succinate, respectively. Fumarate inhibits the TETs and HIF-P4Hs, leading to a reduction in global 5-hmC levels and HIF-1α/HIF-2α stabilization, respectively. Expression of the glycolysis-associated HIF1 target genes PGK1, HK2, and ENO1 was reduced in cells treated with fumarate, suggesting that the TETs and 5-hmC have a crucial role in the regulation of these genes. The expression of VEGFA and BNIP3 mRNAs was nevertheless, increased in cells treated with fumarate, suggesting that these genes are more driven by HIF-1α/HIF-2α stabilization than by the reduction in 5-hmC levels. *, cells treated with succinate showed reductions in VEGFA, PGK1, and HK2 mRNA expression, suggesting that the TETs and 5-hmC can regulate their expression when HIF-1α is not stabilized. HIF target genes are thus regulated in a multilayered manner in which 5-hmC acts as an additional layer of regulation. Not all HIF target genes are regulated equally by 5-hmC, suggesting that there could be promoter-specific gains and reductions in 5-hmC via TETs.