Hypothesis

Aging‑associated epigenetic remodeling at the CDKN2A/B locus does not merely reflect senescence but actively contributes to autophagy suppression by altering the chromatin state of regulators of the mTORC1‑TFEB axis. We propose that loss of repressive H3K27me3 and gain of activating marks at CDKN2A/B promoters facilitate transcription of a non‑coding RNA or enhancer RNA that sequesters the lysosomal calcium channel MCOLN1, blunting the calcium‑dependent activation of calcineurin‑TFEB signaling. Consequently, TFEB remains phosphorylated and cytosolic, autophagy genes are not transcribed, and lysosomal biogenesis declines. This model positions CDKN2A/B epigenetic changes upstream of mTORC1 hyperactivity, providing a chromatin‑mediated link that explains why autophagy inhibition coincides with p16INK4a accumulation.

Mechanistic Model

1. Epigenetic shift – With age, CDKN2A/B promoter loses H3K27me3 (PRC2‑mediated repression) and acquires H3K4me3/H3K27ac, leading to increased transcription of p16INK4a and a nearby enhancer‑derived lncRNA (e.g., ANRIL‑like) https://pmc.ncbi.nlm.nih.gov/articles/PMC6743916/.
2. lncRNA‑mediated calcium buffering – The lncRNA binds to MCOLN1 (TRPML1) or associated regulators, reducing lysosomal Ca2+ release upon autophagic stress https://doi.org/10.1038/ncomms14995.
3. Impaired calcineurin‑TFEB activation – Lower lysosomal Ca2+ diminishes calcineurin activity, preventing TFEB dephosphorylation and nuclear translocation, even when mTORC1 signaling is modestly active https://www.imrpress.com/journal/FBL/30/9/10.31083/FBL38730.
4. Feedback loop – Cytosolic TFEB fails to induce lysosomal biogenesis genes, further decreasing lysosomal Ca2+ stores and reinforcing autophagy suppression.
5. p16INK4a accumulation – Because autophagy is inhibited, p16INK4a protein is not degraded, reinforcing the senescence phenotype https://pmc.ncbi.nlm.nih.gov/articles/PMC7370706/.

Predictions & Experiments

• Prediction 1: In aged mouse liver or muscle, nuclei sorted for high H3K27ac at CDKN2A/B will show reduced MCOLN1 mRNA or protein levels and lower lysosomal Ca2+ flux compared with low‑epigenetic‑activity nuclei. Test by CUT&Tag for H3K27ac, followed by RNA‑seq and Lysosomal Ca2+ imaging with GCaMP‑ML.
• Prediction 2: Knock‑down of the CDKN2A/B‑associated lncRNA (using antisense oligos) in aged primary fibroblasts will restore lysosomal Ca2+ release, increase TFEB nuclear localization, and boost LC3‑II turnover without altering mTORC1 activity (measured by phospho‑S6K).
• Prediction 3: Pharmacologic activation of MCOLN1 (e.g., with ML‑SA1) in aged cells should bypass the epigenetic block, rescuing autophagy flux even when CDKN2A/B remains hypomethylated.
• Falsification: If manipulating CDKN2A/B epigenetics or its lncRNA does not affect lysosomal Ca2+ or TFEB localization, the model is refuted.

Potential Implications

Linking a senescence‑associated epigenetic locus to lysosomal calcium signaling offers a mechanistic bridge between two hallmarks of aging: epigenetic dysregulation and loss of proteostasis. It suggests that epigenetic therapies targeting CDKN2A/B (e.g., EZH2 inhibitors or CRISPR‑based repressors) could re‑activate autophagy independently of mTOR inhibition, potentially reducing the need for chronic rapamycin treatment and its side effects.

References

• CDKN2A promoter methylation decreases with age: https://pmc.ncbi.nlm.nih.gov/articles/PMC6743916/
• Maintenance of quiescence requires active repression at the INK4a/CDKN2A locus: https://doi.org/10.4161/15384101.2014.965072
• Blocking autophagy causes p16 accumulation without altering p16 transcription: https://pmc.ncbi.nlm.nih.gov/articles/PMC7370706/
• Age-related autophagy decline primarily attributed to mTORC1 hyperactivation impairing TFEB: https://www.imrpress.com/journal/FBL/30/9/10.31083/FBL38730
• PRC2-centered signature defines age-associated hypermethylation and gene expression changes: https://doi.org/10.1080/15592294.2015.1040619
• Lysosomal calcium signaling and autophagy: https://doi.org/10.1038/ncomms14995