Mij
Senior Member (Voting Rights)
Editors Summary
Alpha-ketoglutarate (αKG) is a metabolite used by enzymes to chemically modify chromatin, which ensures appropriate gene regulation. However, how αKG is supplied to these enzymes in the nucleus is unclear. Sternisha et al. transferred an αKG-sensing transcription factor from bacteria to human cells to monitor nuclear αKG. They found that αKG produced by glutamate pyruvate transaminase 2 in mitochondria is a key source of αKG that is shuttled to nuclei. Loss-of-function mutations in this enzyme lead to dysregulated chromatin structure and misexpression of genes that are required for brain development. —Stella M. Hurtley
Structured Abstract
Introduction
The metabolite α-ketoglutarate (αKG) is a substrate for dioxygenase enzymes that demethylate histones and DNA in the nucleus, influencing gene expression, cell fate, and tumor suppression. Despite the importance of nuclear αKG for chromatin-modifying enzyme function, mechanisms that govern this metabolite pool remain poorly defined. Revealing these mechanisms is vital for understanding how appropriate chromatin structure and gene expression patterns are maintained under homeostatic conditions and dysregulated in disease.
Rationale
Deciphering how nuclear αKG abundance is regulated in human cells has been challenging for several reasons. First, methods developed to study compartmentalized metabolism in mitochondria, lysosomes, and other organelles are not readily transferrable to nuclei. Second, intracellular αKG is regulated by a complex network of more than 100 enzymes and transporters that act directly on this metabolite. To address these challenges, we sought to create a biosensor capable of detecting changes in nuclear αKG and use it to identify the molecular processes that govern this metabolite pool.
Results
We engineered the transcriptional “αKG-ON biosensor system” by leveraging NtcA, an αKG-responsive cyanobacterial transcription factor. Chimeric NtcA proteins were expressed together with a fluorescent reporter gene driven by a synthetic promoter featuring NtcA binding sites derived from cyanobacterial genomes. Together, these elements enabled fluorescence-based monitoring of the nuclear αKG pool in living human cells. We used the αKG-ON biosensor system in a forward genetic screen to identify genes that play dominant roles in controlling αKG abundance in nuclei. This approach uncovered an interorganelle pathway that mediates the transfer of αKG from mitochondria to nuclei. Within this pathway, mitochondrial glutamic-pyruvic transaminase 2 (GPT2) synthesizes αKG that is preferentially exported to the cytosol through the SLC25A11 transporter, a component of the malate-aspartate shuttle. Once in the cytosol, αKG may enter the nucleus or undergo catabolism to glutamate by the branched-chain amino acid transaminase 1 (BCAT1). Our findings align with prior research showing that BCAT1 competes with chromatin demethylating enzymes for access to nucleocytosolic αKG and reveal sequential activities of GPT2 and SLC25A11 as key suppliers of this metabolite pool.
Disrupting this interorganelle pathway of αKG metabolism caused chromatin hypermethylation in cultured cells in vitro and in tissues in vivo. A mouse model of GPT2 deficiency, an inborn error of metabolism caused by inactivating mutations in GPT2, displayed increases in methylation of both DNA and histones in brain tissue coupled with profound dysregulation of neurodevelopmental gene expression programs. Administering an esterified form of αKG to Gpt2-null mice restored chromatin methylation patterns, normalized gene expression, and improved mouse fitness, establishing a causal link between nuclear αKG depletion and the molecular pathogenesis of GPT2 deficiency.
Conclusion
This study provides a tool for monitoring αKG abundance in the nuclei of human cells and defines a pathway for mitochondrion-to-nucleus transfer of αKG that sustains epigenomic regulation. These findings underscore compartmentalized metabolism as a critical determinant of epigenetic state and reveal nuclear αKG depletion as a driver of neurodevelopmental defects in GPT2 deficiency. Moreover, our work suggests that αKG supplementation represents a potential strategy to counteract pathogenic mechanisms in patients with this disorder.
STUDY
Alpha-ketoglutarate (αKG) is a metabolite used by enzymes to chemically modify chromatin, which ensures appropriate gene regulation. However, how αKG is supplied to these enzymes in the nucleus is unclear. Sternisha et al. transferred an αKG-sensing transcription factor from bacteria to human cells to monitor nuclear αKG. They found that αKG produced by glutamate pyruvate transaminase 2 in mitochondria is a key source of αKG that is shuttled to nuclei. Loss-of-function mutations in this enzyme lead to dysregulated chromatin structure and misexpression of genes that are required for brain development. —Stella M. Hurtley
Structured Abstract
Introduction
The metabolite α-ketoglutarate (αKG) is a substrate for dioxygenase enzymes that demethylate histones and DNA in the nucleus, influencing gene expression, cell fate, and tumor suppression. Despite the importance of nuclear αKG for chromatin-modifying enzyme function, mechanisms that govern this metabolite pool remain poorly defined. Revealing these mechanisms is vital for understanding how appropriate chromatin structure and gene expression patterns are maintained under homeostatic conditions and dysregulated in disease.
Rationale
Deciphering how nuclear αKG abundance is regulated in human cells has been challenging for several reasons. First, methods developed to study compartmentalized metabolism in mitochondria, lysosomes, and other organelles are not readily transferrable to nuclei. Second, intracellular αKG is regulated by a complex network of more than 100 enzymes and transporters that act directly on this metabolite. To address these challenges, we sought to create a biosensor capable of detecting changes in nuclear αKG and use it to identify the molecular processes that govern this metabolite pool.
Results
We engineered the transcriptional “αKG-ON biosensor system” by leveraging NtcA, an αKG-responsive cyanobacterial transcription factor. Chimeric NtcA proteins were expressed together with a fluorescent reporter gene driven by a synthetic promoter featuring NtcA binding sites derived from cyanobacterial genomes. Together, these elements enabled fluorescence-based monitoring of the nuclear αKG pool in living human cells. We used the αKG-ON biosensor system in a forward genetic screen to identify genes that play dominant roles in controlling αKG abundance in nuclei. This approach uncovered an interorganelle pathway that mediates the transfer of αKG from mitochondria to nuclei. Within this pathway, mitochondrial glutamic-pyruvic transaminase 2 (GPT2) synthesizes αKG that is preferentially exported to the cytosol through the SLC25A11 transporter, a component of the malate-aspartate shuttle. Once in the cytosol, αKG may enter the nucleus or undergo catabolism to glutamate by the branched-chain amino acid transaminase 1 (BCAT1). Our findings align with prior research showing that BCAT1 competes with chromatin demethylating enzymes for access to nucleocytosolic αKG and reveal sequential activities of GPT2 and SLC25A11 as key suppliers of this metabolite pool.
Disrupting this interorganelle pathway of αKG metabolism caused chromatin hypermethylation in cultured cells in vitro and in tissues in vivo. A mouse model of GPT2 deficiency, an inborn error of metabolism caused by inactivating mutations in GPT2, displayed increases in methylation of both DNA and histones in brain tissue coupled with profound dysregulation of neurodevelopmental gene expression programs. Administering an esterified form of αKG to Gpt2-null mice restored chromatin methylation patterns, normalized gene expression, and improved mouse fitness, establishing a causal link between nuclear αKG depletion and the molecular pathogenesis of GPT2 deficiency.
Conclusion
This study provides a tool for monitoring αKG abundance in the nuclei of human cells and defines a pathway for mitochondrion-to-nucleus transfer of αKG that sustains epigenomic regulation. These findings underscore compartmentalized metabolism as a critical determinant of epigenetic state and reveal nuclear αKG depletion as a driver of neurodevelopmental defects in GPT2 deficiency. Moreover, our work suggests that αKG supplementation represents a potential strategy to counteract pathogenic mechanisms in patients with this disorder.
STUDY