Hypothesis

Nuclear acetyl-CoA produced by ACSS2 promotes the liquid-liquid phase separation (LLPS) of the histone acetyltransferase p300/CBP at super-enhancers of autophagy and neuroprotective genes. This acetylation‑dependent condensate formation concentrates p300/CBP activity, boosts histone acetylation at target loci, and sustains a transcriptional program that opposes senescence. When nuclear acetyl-CoA declines, p300/CBP remains diffuse, loses enhancer‑specific activity, and shifts toward acetylation of inflammatory loci, thereby driving age‑related dysfunction.

Mechanistic Rationale

1. Acetylation of low‑complexity domains (LCDs) regulates LLPS

   • p300/CBP contain LCDs enriched in lysine residues that, when acetylated, reduce positive charge and increase propensity for multivalent weak interactions, a principle demonstrated for other transcriptional coactivators {Spatiotemporal control of acetyl-CoA metabolism in chromatin regulation}.
   • Nuclear acetyl-CoA directly fuels these acetylation events; thus, local acetyl-CoA concentration sets the threshold for condensate formation.

2. Spatial targeting by ACSS2 creates epigenomic hotspots

   • ACSS2 is recruited to specific chromatin regions via interaction with lamin B1 and CTCF, generating microscales of high acetyl-CoA that nucleate p300/CBP droplets preferentially at lamina‑associated domains (LADs) housing autophagy gene clusters {ACSS2 upregulation enhances neuronal resilience to aging and tau-associated neurodegeneration}.
   • This explains why boosting nuclear acetyl-CoA yields gene‑specific effects rather than global histone hyperacetylation.

3. Phase‑separated p300/CBP enhances TFEB‑driven transcription

   • Within condensates, p300/CBP acetylates both histones and TFEB, increasing TFEB nuclear retention and transcriptional activity on lysosomal and autophagy genes {Acetyl-CoA Metabolism and Histone Acetylation in the Regulation of Aging and Lifespan}.
   • The condensate also excludes HDACs, creating a locally deacetylated‑free zone that sustains high acetylation levels.

4. Shift to diffuse p300/CBP promotes senescence-associated secretory phenotype (SASP)

   • When acetyl-CoA falls, p300/CBP diffuses, acetylates nucleosomes at NF‑κB‑regulated promoters, and cooperates with BRD4 to sustain inflammatory gene expression {Epigenetic Regulation of Aging and its Rejuvenation}.
   • This provides a mechanistic link between metabolic decline and the inflammatory arm of aging.

Testable Predictions

• Prediction 1: Neuronal overexpression of an acetylation‑deficient p300/CBP LCD mutant (K→R) will abolish the lifespan‑extending effect of ACSS2 overexpression, despite normal nuclear acetyl-CoA levels.
• Prediction 2: Acute treatment with 1,6‑hexanediol (an LLPS disruptor) will phenocopy ACSS2 knockdown, reducing histone H3K27ac at autophagy gene enhancers and increasing SASP markers in cultured astrocytes.
• Prediction 3: Fluorescence recovery after photobleaching (FRAP) of p300/CBP‑GFP will show slower recovery (indicative of condensate formation) in young neurons with high ACSS2, and faster recovery in aged neurons or after ACSS2 inhibition.
• Prediction 4: Chromatin immunoprecipitation followed by sequencing (ChIP‑seq) for p300/CBP in conditions of high vs. low nuclear acetyl-CoA will reveal a shift from autophagy‑gene super-enhancers to NF‑κB‑bound inflammatory loci.

Experimental Approach

1. Manipulate nuclear acetyl-CoA

   • Use AAV‑mediated neuron‑specific ACSS2 overexpression or CRISPRi knockdown in mouse models.
   • Complement with pharmacologic modulation of ACSS2 activity (e.g., using the inhibitor VY‑3‑135).

2. Assess LLPS behavior

   • Generate p300/CBP‑GFP constructs with wild‑type or LCD acetylation‑site mutants.
   • Perform live‑cell FRAP and fluorescence correlation spectroscopy in primary neurons and glial cultures.

3. Measure transcriptional output

   • Conduct RNA‑seq and ATAC‑seq to identify changes in autophagy versus SASP pathways.
   • Validate key targets (TFEB, LC3B, IL6, CXCL10) by qPCR and Western blot.

4. Functional readouts

   • Evaluate lysosomal activity (LysoTracker), autophagic flux (LC3‑II/p62 turnover), and cognitive performance (Morris water maze) in aged mice.
   • Assess tissue‑specific impact by repeating experiments in liver and muscle to probe the ACSS2 paradox.

Potential Outcomes and Implications

If the hypothesis holds, it will establish a direct mechanistic bridge linking a metabolic metabolite (nuclear acetyl-CoA) to the biophysical state of a chromatin regulator, explaining how localized acetyl-CoA pools can exert gene‑specific effects while avoiding global acetylation noise. This reframing suggests that therapeutic strategies aiming to enhance longevity should focus not only on raising acetyl-CoA levels but also on promoting or stabilizing the phase‑separated state of p300/CBP (e.g., via small‑molecule modulators of LCD interactions). Conversely, agents that disrupt pathologic condensates (as explored in neurodegenerative disease) might inadvertently accelerate aging if they impair beneficial p300/CBP LLPS at autophagy loci. Testing these ideas will clarify whether nuclear acetyl-CoA’s anti‑aging power is mediated through concentration‑dependent phase separation, offering a novel axis for intervention.