mTORC1-Dependent Epigenetic Silencing of PAX6 Drives Limbal Stem Cell Aging

Hypothesis

The IIS/IGF-1/mTORC1 signaling axis directly suppresses PAX6 transcription in limbal epithelial stem cells (LESCs) through mTORC1‑dependent phosphorylation of the histone acetyltransferase p300, reducing its activity at the PAX6 promoter and leading to epigenetic silencing. This positions mTORC1 as an upstream controller that couples nutrient sensing to the age‑related decline of PAX6, thereby driving the accumulation of dysfunctional LESC‑like cells.

Mechanistic Rationale

• mTORC1 activates S6K1, which can phosphorylate p300 on serine residues known to diminish its acetyltransferase activity (see [3] for mTORC1‑S6K1 signaling breadth).
• Reduced p300 activity lowers H3K27ac at the PAX6 enhancer, establishing a repressive chromatin state that diminishes PAX6 mRNA despite unchanged transcription factor availability.
• PAX6 loss then triggers stress signatures and impaired differentiation, as shown in Pax6‑deficient mice ([1]).

Thus, PAX6 decline is not merely a downstream marker but a consequential effector of mTORC1‑mediated epigenetic reprogramming.

Testable Predictions

1. Pharmacologic inhibition of mTORC1 (e.g., rapamycin or torin1) in aged mouse limbus will increase p300 acetylation, restore H3K27ac at the PAX6 locus, elevate PAX6 expression, and rescue LESC proliferative and differentiation capacity.
2. Genetic activation of mTORC1 in young LESCs (via Rheb overexpression or TSC1 knockout) will recapitulate the epigenetic changes (decreased p300 activity, loss of H3K27ac) and cause PAX6 down‑regulation together with aging‑like phenotypes.
3. CRISPR‑mediated disruption of the S6K1 phosphorylation sites on p300 will block mTORC1‑dependent PAX6 silencing, rendering LESC resistant to rapamycin‑insensitive aging signals.

Experimental Approach

• In vivo: Treat 20‑month‑old mice with rapamycin (10 mg/kg, i.p., three times weekly) for 8 weeks; isolate limbal epithelium; perform ChIP‑qPCR for H3K27ac and p300 at the PAX6 enhancer, RT‑qPCR and immunofluorescence for PAX6, and colony‑forming efficiency assays.
• Ex vivo: Culture human LESCs from young donors; transfect with constitutively active Rheb; assess p300 phosphorylation (phospho‑specific antibody), H3K27ac ChIP, PAX6 mRNA/protein, and differentiation markers (K3, K12).
• Genetic: Generate Pax6‑fl;K12‑CreERT2 mice with inducible TSC1 deletion in the limbus; monitor PAX6 loss and functional decline over time.

Falsifiability

If mTORC1 inhibition fails to increase p300 activity, H3K27ac, or PAX6 levels in aged limbus, or if forced mTORC1 activation does not reduce PAX6, the hypothesis is refuted. Conversely, confirmation would support a unified upstream controller (mTORC1) acting through an epigenetic hub (p300) to drive a key aging hallmark (PAX6 loss) in a specific stem cell niche.