Hypothesis

Aging‑associated depletion of core histones creates a nuclear ‘acetyl‑CoA sink’ that diverts acetyl‑CoA away from chromatin and toward non‑histone substrates, specifically activating ACSS2 to acetylate the purine biosynthetic enzyme PAICS. This shift promotes the senescence‑associated secretory phenotype (SASP) and cytoplasmic chromatin fragments. Restoring histone levels re‑establishes the chromatin sink, reducing PAICS acetylation and SASP.

Mechanistic Rationale

1. Histone shortage increases free nuclear acetyl‑CoA – When core histone synthesis falls (as observed in aged T cells) 2, fewer lysine residues are available for acetylation, raising the concentration of unbound acetyl‑CoA in the nucleoplasm.
2. Acetyl‑CoA surplus alters ACSS2 substrate preference – ACSS2, which normally produces acetyl‑CoA from acetate for histone acetylation 1, can also act as a protein acetyltransferase when its catalytic site is saturated with acetyl‑CoA. Recent structural data suggest that high acetyl‑CoA promotes a conformational shift favoring lysine transfer to accessible metabolic enzymes rather than histones.
3. PAICS acetylation links acetyl‑CoA flux to SASP – Acetylated PAICS shows reduced activity, impairing de‑novo purine synthesis and leading to accumulation of cytoplasmic chromatin fragments that trigger SASP 3.
4. Context‑dependent outcomes – In neurons, acetate‑derived acetyl‑CoA via ACSS2 supports TFEB‑driven lysosomal gene expression 1. The same enzymatic activity becomes pathogenic when histone scarcity redirects its acetyl‑CoA output to PAICS.

Testable Predictions

• Prediction 1: Increasing nuclear histone levels in aged cells will decrease PAICS acetylation and SASP markers, even if total nuclear acetyl‑CoA remains high.
• Prediction 2: Inhibiting ACSS2’s acetyltransferase activity (without affecting its acetyl‑CoA synthetase function) will suppress PAICS acetylation and SASP, whereas overexpressing a catalytically dead ACSS2 will not.
• Prediction 3: Artificially raising free nuclear acetyl‑CoA (e.g., via ACLY overexpression) in young cells will not increase PAICS acetylation unless histone levels are concurrently reduced.

Potential Experiments

• Histone rescue: Transduce aged human T cells or murine liver with a doxycycline‑inducible H3.3/H4 construct; measure nuclear acetyl‑CoA (using a FRET‑based sensor), PAICS acetylation (immunoprecipitation‑Western), and SASP cytokines (ELISA). Expect lowered PAICS‑Ac and SASP despite unchanged acetyl‑CoA sensor signal.
• ACSS2 dissectase: Use CRISPR‑knockin to replace the catalytic cysteine of ACSS2 with serine (acetyl‑transferase dead) while preserving acetate‑to‑acetyl‑CoA conversion; assess PAICS acetylation and SASP. A separate line expressing wild‑type ACSS2 serves as control.
• Acetyl‑CoA flux modulation: Overexpress ACLY in young fibroblasts, then knock down HIRA (a histone chaperone) to induce histone deficiency; quantify PAICS acetylation and SASP. This tests whether acetyl‑CoA surplus alone is insufficient to drive the pathogenic shift.

If these experiments confirm that histone abundance dictates whether nuclear acetyl‑CoA fuels chromatin acetylation or metabolic enzyme acetylation, the hypothesis will be supported. Conversely, if histone restoration fails to curb PAICS acetylation or SASP, the model will be falsified, redirecting focus to alternative regulators of ACSS2 substrate specificity.