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.
Highlights
The 2-oxoglutarate-dependent dioxygenases (2-OGDDs)2 comprise an enzyme family of about 70 members in humans [1, 2]
We showed in cellulo that fumarate and succinate play a role in the regulation of certain hypoxia-inducible factor (HIF) target genes via translocation 5-methylcytosine dioxygenases (TETs) inhibition, suggesting that 5-hmC has a role in regulation of the hypoxia response
We determined the Km values of Tets 1 and 2 for molecular oxygen and show that their activity is not highly dependent on oxygen, consistent with these enzymes remaining catalytically active when induced by hypoxia [13]
Summary
Expression and Purification of Recombinant Enzymes—The catalytic domains for murine Tets 1–3 in the pFasbac-HTb vector with a N-terminal FLAG tag were a gift from Dr Y. The activity assays were carried out in a final volume of 50 l and the reaction mixture contained 2 g/l of bovine serum albumin, 50 mM Tris (pH 7.8), 0.1 mM DTT, 5 mM ascorbate, 0.05 mM FeSO4, 0.6 mM 2-oxo[1-14C]glutarate, and 1.8 M DNA substrate. Determination of Intranuclear and Cytosolic Succinate Concentrations following DMS Treatment—Nuclear and cytosolic fractions were extracted from the cells using NEPER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific). Quantification of 5-mC and 5-hmC by High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry—Genomic DNA was extracted from the cells by lysing the cells with a buffer containing 0.1 M Tris (pH 8.5), 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, and 0.1 mg/ml of Proteinase K and subjecting the lysates for phenol-chloroform extraction. All data are shown as mean Ϯ S.E. except for the enzyme kinetics and intranuclear and cytosolic succinate concentrations, which are shown as mean Ϯ S.D. (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.005)
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