Hypothesis: Hierarchical autophagic degradation of PRC2 subunits drives age‑dependent CDKN2A/B derepression and senescence

Mechanistic rationale

Aging cells show progressive loss of Polycomb Repressive Complex 2 (PRC2) mediated H3K27me3 at the CDKN2A/B locus, leading to p16^INK4a upregulation 12. While bulk autophagy declines with age, selective autophagy remains active and can target specific protein complexes via receptors such as p62/SQSTM1 3. We hypothesize that, under nutrient stress, the autophagic machinery preferentially engages PRC2 core subunits (EZH2, SUZ12, EED) in a defined order, stripping the complex from chromatin and triggering CDKN2A/B activation. This ordered depletion mirrors a triage mechanism where the cell sacrifices epigenetic silencing to initiate a senescence program that limits proliferation of damaged cells.

Novel mechanistic insight

PRC2 subunits contain lysine‑rich regions that become ubiquitinated in response to oxidative stress, creating a recognition motif for p62. Unlike random cargo, ubiquitinated EZH2 is modified first because its SET domain exposes lysine residues that are preferentially modified by the E3 ligase TRIM28 under low‑ATP conditions. Subsequent ubiquitination of SUZ12 and EED follows a kinetic hierarchy dictated by their accessibility within the complex. Once tagged, p62 recruits LC3‑positive phagophores, leading to sequential autophagic degradation. The loss of EZH2 first reduces H3K27me3 nucleation, SUZ12 removal destabilizes the remaining complex, and EED elimination blocks allosteric activation, collectively erasing repressive marks and permitting transcription factors (e.g., NF‑κB) to bind the CDKN2A/B promoter.

Testable predictions

1. In primary human fibroblasts subjected to serum starvation, EZH2 ubiquitination peaks before SUZ12 and EED, detectable by immunoprecipitation followed by ubiquitin‑specific blotting.
2. Knock‑down of p62 abolishes the ordered loss of PRC2 subunits and delays p16^INK4a induction despite sustained autophagy flux.
3. Overexpression of a non‑ubiquitinatable EZH2 lysine‑mutant preserves H3K27me3 at CDKN2A/B and reduces senescence markers under stress.
4. Pharmacological inhibition of autophagosome formation (e.g., with VPS34 inhibitor) blocks PRC2 degradation but does not affect bulk proteasome activity, distinguishing the selective route.

Experimental approach

• Step 1: Treat young and old mouse muscle satellite cells with EBSS to induce autophagy; isolate chromatin fractions at 0, 2, 4, 6 h.
• Step 2: Perform sequential western blots for EZH2, SUZ12, EED, and p62‑LC3 colocalization via immunofluorescence.
• Step 3: Use CRISPR‑edited EZH2 K→R mutants to test ubiquitination dependence.
• Step 4: Measure CDKN2A/B mRNA (qPCR) and p16 protein (flow cytometry) alongside senescence‑associated β‑galactosidase.
• Step 5: Conduct rescue experiments with lysosomal protease inhibitor (chloroquine) to confirm autophagic degradation.

Potential implications

If validated, this model redefines autophagy not merely as a cleanup pathway but as an epigenetic timer that actively shapes the transcriptional landscape of aging. It suggests that modulating the selectivity of autophagic receptors could uncouple harmful senescence from beneficial quality control, offering a lever to extend healthspan without compromising protein homeostasis.