Chronic JNK signaling in senescent cells shifts AP-1 dimer composition from transient c-Jun/c-Fos heterodimers to stable Fra2/JunD complexes that sustain SASP expression. This switch is driven by the senescence‑specific histone variant H2A.J, which remodels nucleosomes at inflammatory promoters to create high‑affinity docking sites for Fra2/JunD while excluding c-Jun/c-Fos. Once Fra2/JunD binds, it recruits p300/CBP acetyltransferase activity, reinforcing an open chromatin state that further amplifies mitochondrial ROS production through NF‑κB‑dependent transcription of NADPH oxidase subunits. The resulting ROS feeds back to activate JNK, establishing a self‑reinforcing loop that locks the SASP in a constitutively active state.

Key predictions:

1. In primary human fibroblasts rendered senescent by ionizing radiation, ChIP‑seq for Fra2 and JunD will show increased occupancy at IL6, IL8, and CXCL1 promoters concomitant with reduced c‑Jun/c‑Fos binding, whereas pre‑senescent cells will display the opposite pattern.
2. Knockdown of H2A.J using CRISPRi will prevent Fra2/JunD recruitment without affecting total JNK activity, leading to a selective loss of SASP cytokines while preserving p21‑mediated cell‑cycle arrest.
3. Pharmacological inhibition of JNK3 (but not JNK1/2) with a selective peptide inhibitor will reduce Fra2 phosphorylation, diminish Fra2/JunD DNA binding, and attenuate SASP secretion, whereas JNK1/2 inhibition will accelerate senescence entry as previously reported.
4. Overexpression of a Fra2 mutant lacking the JNK phosphorylation site (Ser325A) will fail to sustain SASP despite persistent mitochondrial ROS, confirming that JNK‑mediated Fra2 phosphorylation is required for the chronic dimer state.

Experimental approach:

• Generate isogenic IMR‑90 lines expressing inducible H2A.J shRNA, Fra2‑WT, Fra2‑S325A, and JunD‑WT constructs.
• Treat cells with low‑dose rotenone to elevate mitochondrial ROS and measure SASP by multiplex ELISA at 0, 3, 7, and 14 days.
• Perform sequential ChIP (Re‑ChIP) for Fra2 followed by JunD to confirm dimer formation on target promoters.
• Assess chromatin accessibility via ATAC‑seq and histone acetylation (H3K27ac) to link H2A.J deposition to enhancer activation.
• Use live‑cell MitoSOX imaging to correlate ROS fluctuations with JNK activity (phospho‑JNK immunoblot) and AP‑1 dimer composition (phospho‑specific antibodies).

Falsifiability: If H2A.J depletion does not alter Fra2/JunD promoter occupancy or SASP levels, or if JNK3 inhibition fails to reduce Fra2 phosphorylation and SASP while leaving senescence markers unchanged, the proposed loop would be refuted. Likewise, demonstrating that c‑Jun/c‑Fos remains the dominant AP-1 dimer in late‑stage senescent cells under these conditions would invalidate the hypothesis of a dimer switch driving chronic inflammaging.