The TET2 DNA dioxygenase regulates cell identity and suppresses tumorigenesis by modulating DNA methylation and expression of a large number of genes. How TET2, like most other chromatin modifying enzymes, is recruited to specific genomic sites is unknown. Here we report that WT1, a sequence-specific transcription factor, is mutated in a mutually exclusive manner with TET2, IDH1 and IDH2 in acute myeloid leukemia (AML). WT1 physically interacts with and recruits TET2 to its target genes to activate their expression. The interaction between WT1 and TET2 is disrupted by multiple AML-derived TET2 mutations. TET2 suppresses leukemia cell proliferation and colony formation in a manner dependent on WT1. These results provide a mechanism for targeting TET2 to specific DNA sequence in the genome. Our results also provide an explanation for the mutual exclusivity of WT1 and TET2 mutations in AML and suggest an IDH1/2-TET2-WT1 pathway in suppressing AML.

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose-6-phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH-producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS. SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5-dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.

The immune-response gene 1 (IRG1) plays a key role in anti-pathogen defense, as deletion of Irg1 in mice causes severe defects in response to bacterial and viral infection, and decreased survival 1, 2 . IRG1 transcription is rapidly induced by pathogen infection and in ammatory conditions primarily in cells of myeloid lineage 3 . IRG1 encodes a mitochondrial metabolic enzyme, aconitate decarboxylase 1 (ACOD1), that catalyzes the decarboxylation of cis-aconitate to produce the anti-in ammatory metabolite itaconic acid (ITA) 4 . Several molecular processes are affected by ITA, including succinate dehydrogenase (SDH) inhibition 5 , resulting in succinate accumulation and metabolic reprogramming 6,7 , and alkylation of protein cysteine residues, inducing the electrophilic stress response mediated by NRF2 and IκBζ 8, 9 and impairing aerobic glycolysis 10 . However, the mechanisms by which ITA exerts its profound antiin ammatory effect still remains to be fully elucidated. Here, we show that ITA is a potent inhibitor of the TET family DNA dioxygenases, which catalyze the conversion of 5-methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC) during the process of active DNA demethylation. ITA binds to the same site of α-ketoglutarate (α-KG) in TET2, inhibiting its catalytic activity. Lipopolysaccharides (LPS) treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals TET2 is a major target of ITA in suppressing LPS-induced genes, including those regulated by NF-κB and STAT signaling pathways. In vivo, ITA decreases 5hmC, reduces LPS-induced acute pulmonary edema and lung and liver injury, and protects mice against lethal endotoxaemia in a manner that is dependent on the catalytic activity of Tet2. Our study thus identi es ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the in ammatory response. MainDeletion of the Irg1 gene or treatment with cell permeable ITA alters the transcriptional signature in response to LPS 2 . We speculated that ITA may impact epigenetics to in uence gene expression, and therefore, we determined the effect of Irg1 expression and ITA accumulation on global histone and DNA de/methylation in transfected HEK293T cells (Extended Data Fig. 1a). We found that ectopic expression of either wild-type or catalytic inactive mutant Irg1 had little effect on mono-, di-, and trimethylation of all ve histone H3 lysine residues (Extended Data Fig. 1b, 1c). In contrast, expression of wild-type Irg1, but not the catalytic inactive mutant, dramatically reduced Tet2-mediated global 5hmC in cells (Fig. 1a and Extended Data Fig. 1d-e). Like α-KG, which is a crucial co-substrate for the activity of TET2, ITA is also a dicarboxylic acid containing a 4-or 5-carboxylate that, in the case of α-KG, forms hydrogen and ionic bonds with H1416, R1896, and S1898 in TET2 11 . Of note, α-KG binds to Fe(II) in a bidentate manner via its C-1 carboxylate and C-2 keto groups, which are lacking in ITA. This raises the possibi...