Mitochondrial L-2-hydroxyglutarate is a physiologic signaling metabolite [ChIP-seq]

L-2-hydroxyglutarate (L-2-HG) is a low-abundance metabolite in mammals, due to the mitochondrial enzyme L-2-HG dehydrogenase (L2HGDH), which oxidizes L-2-HG to 2-oxoglutarate (2-OG) to prevent its accumulation1. In humans lacking L2HGDH activity, L-2-HG builds up, leading to L-2-HG aciduria, a rare childhood neurometabolic disorder2. Thus, L-2-HG is classified as a toxic metabolite2–5. Furthermore, L-2-HG is produced in response to hypoxia, acidic pH, and electron transport chain (ETC) impairment6–10. We investigated the regulation of L-2-HG levels, its impact on gene expression, and whether it has a physiological role in mice. Here, we report that elevated mitochondrial NADH/NAD+ triggers malate dehydrogenase 2 (MDH2) to produce L-2-HG from 2-OG. By contrast, L2HGDH converts L-2-HG to 2-OG in the mitochondrial matrix without requiring a functional ETC. Nascent gene expression analysis in mouse embryonic stem cells (mESCs) demonstrated that the majority of genes were downregulated by elevated L-2-HG levels. To identify proteins involved in L-2-HG-dependent gene regulation, we used proteome integral solubility alteration assays (PISA), which showed that L-2-HG engages the RNA demethylase FTO and H3K9 demethylases KDM4A/B/C in mESC nuclear extracts. The L-2-HG-dependent accumulation of m6A in mRNAs and H3K9me3 at the gene loci negatively correlated with gene expression. Importantly, mitochondrial L2HGDH overexpression during early embryogenesis lowered basal L-2-HG levels and resulted in viable mice at birth but triggered reduced growth, impaired kidney function, and post-natal mortality. Thus, L-2-HG primarily downregulates gene expression and is a signaling metabolite necessary for post-natal mammalian growth and survival. These findings suggest that other previously considered toxic metabolites might also play physiological roles.