Low α‑KG‑Dependent Stabilization of a PRC2‑Guiding lncRNA Drives Epigenetic Silencing of Autophagy Genes in Aging

Aging cells actively suppress autophagy by locking autophagy gene promoters in a repressive chromatin state through a metabolite‑sensitive lncRNA that recruits PRC2.

The age‑associated drop in SLC1A5‑mediated glutamine import lowers intracellular α‑ketoglutarate (α‑KG) levels. α‑KG is a required cofactor for α‑KG‑dependent dioxygenases such as the m6A demethylases ALKBH5 and FTO, which normally destabilize certain nuclear lncRNAs by promoting their degradation. When α‑KG falls, these dioxygenases become less active, leading to the stabilization of lncRNAs that can guide Polycomb Repressive Complex 2 (PRC2) to chromatin. One candidate, MALAT1, is known to interact with EZH2 and to be regulated by m6A methylation; its stability increases when α‑KG‑dependent demethylases are inhibited.

Testable hypothesis: In aged cells, reduced α‑KG stabilizes MALAT1, which then recruits EZH2 to autophagy gene promoters (e.g., TFEB, ULK1, BECN1, ATG7, LC3B), increasing H3K27me3 and silencing transcription. Restoring autophagy flux should be possible by destabilizing MALAT1 or blocking its interaction with EZH2, even without correcting SLC1A5 loss.

Experimental plan:

• Measure SLC1A5 expression, intracellular α‑KG, and MALAT1 levels in young versus aged murine hematopoietic stem cells or skeletal muscle.
• Perform RNA immunoprecipitation (RIP) for EZH2 to quantify MALAT1‑EZH2 binding in both age groups.
• Use CUT&Tag to map H3K27me3 at autophagy gene promoters; predict higher enrichment in aged cells that correlates with MALAT1‑EZH2 occupancy.
• Knockdown MALAT1 with siRNA or antisense oligonucleotides in aged cells and assess autophagy flux via LC3‑II turnover, p62 degradation, and GFP‑LC3 puncta.
• Conversely, overexpress MALAT1 in young cells and test whether it reproduces the aged epigenetic and autophagic phenotype.
• Rescue experiments: supplement aged cells with cell‑permeable α‑KG or overexpress SLC1A5 to see if MALAT1 levels drop and autophagy improves.

Expected outcome: If the hypothesis holds, MALAT1 knockdown (or EZH2 interaction blockade) will reduce H3K27me3 at autophagy promoters and restore flux in aged cells, while α‑KG supplementation will lower MALAT1 stability and produce similar effects. Failure to observe these changes would falsify the model, indicating that α‑KG‑dependent lncRNA stabilization is not the primary route linking metabolic decline to autophagy gene repression.