Hypothesis

In aged cells, reduced glutamine import lowers α‑ketoglutarate (αKG) levels, limiting the activity of the JmjC domain histone demethylase KDM6B. Concurrently, metabolic stress promotes the sequestration of KDM6B into cytoplasmic stress granules through its interaction with RNA‑binding proteins such as G3BP1. This dual hit—diminished enzymatic cofactor availability and physical removal from the nucleus—leads to a local increase of repressive H3K27me3 marks at autophagy‑related promoters (e.g., TFEB, ATG5, LC3B), thereby actively suppressing autophagy gene transcription.

Mechanistic Model

1. Metabolic trigger – Age‑dependent downregulation of SLC1A5 diminishes glutamine influx, decreasing αKG, a required cofactor for KDM6B demethylase activity ([3]).
2. Sequestration trigger – Energetic stress and ROS promote phosphorylation of G3BP1, nucleating stress granules that capture KDM6B via its disordered C‑terminal region, a motif identified in recent proteomic screens of granule components.
3. Epigenetic outcome – Nuclear KDM6B loss permits EZH2‑mediated H3K27me3 deposition at CpG‑rich promoters of autophagy genes, a process already observed for Dram1 ([4]) and reinforced by global PRC2 targeting in aged chromatin ([2]).
4. Feedback loop – Reduced autophagy exacerbates mitochondrial dysfunction, further raising ROS and granule formation, locking the system in a repressed state.

Testable Predictions

• Prediction 1: In hepatocytes from 24‑month‑old mice, nuclear KDM6B levels will be ≥40 % lower than in 3‑month‑old controls, while total cellular KDM6B remains unchanged.
• Prediction 2: ChIP‑qPCR will show a ≥2‑fold increase of H3K27me3 at the TFEB and ATG5 promoters in old tissue, correlating with cytoplasmic stress‑granule markers (G3BP1, TIA‑1).
• Prediction 3: Pharmacological inhibition of stress‑granule assembly (e.g., with ISRIB) or overexpression of a nuclear‑localized KDM6B mutant will rescue H3K27me3 levels and restore autophagy flux (LC3‑II/I ratio) in aged macrophages to youthful levels.

Potential Experiments

• Isolate primary bone‑marrow‑derived macrophages from young and old mice; perform subcellular fractionation followed by western blot for KDM6B and G3BP1 ([1]).
• Conduct CUT&RUN for H3K27me3 and KDM6B at autophagy gene loci; normalize to spike‑in controls.
• Treat aged macrophages with ISRIB (5 µM) for 24 h, then measure autophagic flux using mCherry‑GFP‑LC3 reporter and Seahorse OCR to link granule disruption to metabolic rescue.
• Use CRISPRi to knock down SLC1A5 in young cells to mimic age‑related glutamine loss; assess whether granule formation and H3K27me3 increase recapitulate the aged phenotype.

Falsifiability

If nuclear KDM6B does not decline with age, or if stress‑granule disruption fails to alter H3K27me3 at autophagy promoters despite confirmed granule reduction, the hypothesis would be refuted, pointing to alternative mechanisms such as direct DNA methylation or altered EZH2 activity.

By linking metabolite scarcity, phase‑separation biology, and histone demethylase regulation, this model offers a concrete, reversible node that could explain why autophagy is actively silenced rather than merely worn out in aging tissues.