Redirected nuclear glutamate dehydrogenase supplies Tet3 with alpha-ketoglutarate in neurons

Tet3 is the main alpha -ketoglutarate (alpha KG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How alpha KG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into alpha KG in an NAD(+)-dependent manner, is redirected to the nucleus by the alpha KG-consumer protein Tet3, suggesting on-site production of alpha KG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of alpha KG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity. alpha -ketoglutarate (alpha KG) is an intermediate in the tricarboxylic acid cycle that is required in the nucleus for genomic DNA demethylation by Tet3. Here, the authors show that the enzyme glutamate dehydrogenase, which converts glutamate to alpha KG, is redirected from the mitochondria to the nucleus.Proteomic

5-Formylcytosine to Cytosine Conversion by C-C Bond Cleavage in vivo

Tet enzymes oxidise 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate to dC in the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. The question whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unsolved problem. Here we report the incorporation of synthetic isotope- and (R)-2’-fluorine-labelled dC and fdC-derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labelled fdC probe is efficiently converted into the corresponding labelled dC, most likely after its incorporation into the genome. This allows concluding that fdC is undergoing C-C bond cleavage in stem cells that leads to the direct re-installation of unmodified dC