Hypothesis

Transient autophagy activation during cyclic OSKM expression does not merely supply bulk metabolites; it selectively degrades nuclear lamina components—particularly Lamin B1—to loosen chromatin constraints and facilitate the epigenetic remodeling required for cellular rejuvenation. When autophagy persists beyond the optimal window, continued lamina erosion compromises nuclear integrity, triggering DNA damage responses and senescence.

Mechanistic Insight

Partial reprogramming imposes a sudden demand for nucleosome remodeling and histone exchange, processes that are sterically hindered by a dense lamina meshwork. Autophagy, through its cargo‑recognition machinery (e.g., p62/SQSTM1), can target lamina-associated domains (LADs) for lysosomal degradation when mTORC1 is transiently inhibited by Sox2‑mediated signaling [2]. This selective breakdown releases histone chaperones and remodeling complexes from lamina sequestration, increasing accessibility of pluripotency promoters without requiring global protein turnover [3]. In aged or senescent cells, chronic mTORC1 activity blocks this lamina‑phagy route, locking chromatin in a repressive state [4]. Cyclic OSKM pulses briefly relieve the block, permitting a narrow window of lamina‑dependent autophagy that fuels reprogramming; prolonged activation, however, depletes essential structural nucleoporins and Lamin A/C, leading to nuclear envelope rupture and activation of cGAS‑STING‑driven senescence [6].

Testable Predictions

1. Lamin B1 loss correlates with reprogramming efficiency – Cells undergoing successful partial reprogramming will show a transient (24‑48 h) reduction in Lamin B1 protein levels that precedes OCT4/Nanog upregulation; inhibition of autophagy (Atg5 KO or chloroquine) will abolish this dip and block iPSC colony formation.
2. Lamina‑targeted autophagy reporters – A fluorescent Lamin B1‑GFP‑LC3 fusion will exhibit increased puncta formation specifically during the early phase of cyclic OSKM expression, indicating selective autophagic engulfment of lamina.
3. Rescue by lamina overexpression – Forced expression of an autophagy‑resistant Lamin B1 mutant (lacking the p62‑binding motif) will diminish epigenetic remodeling markers (e.g., H3K27ac at pluripotency enhancers) and reduce reprogramming efficiency, despite normal autophagic flux.
4. Sustained autophagy induces nuclear envelope defects – Extending OSKM expression beyond 5 days will increase γH2AX foci and cytoplasmic DNA accumulation, phenotypes that are mitigated by co‑expressing Lamin A/C or by administering lamin‑stabilizing compounds (e.g., curcumin).

Experimental Design

• Use mouse embryonic fibroblasts (MEFs) with doxycycline‑inducible OSKM.
• Apply 2‑day ON / 2‑day OFF cycles for up to 8 days; collect samples at 0, 24, 48, 72, 96 h after each ON phase.
• Measure Lamin B1, Lamin A/C, and nucleoporin levels by western blot and immunofluorescence; quantify autophagic flux via LC3‑II/p62 turnover.
• Perform AT‑seq and RNA‑seq to assess chromatin accessibility and transcriptional changes at pluripotency loci.
• Include controls: Atg5‑KO MEFs, chloroquine treatment, and overexpression of autophagy‑resistant Lamin B1.
• Assess senescence markers (SA‑β‑gal, p16^INK4a^) and DNA damage (γH2AX) in prolonged‑induction conditions.

If lamina‑specific autophagy is required for the transient increase in chromatin plasticity and its dysregulation leads to senescence, the hypothesis will be supported; failure to observe lamina degradation or its impact on reprogramming will falsify the model.