Hypothesis

Rhythmic phosphorylation of JNK, gated by the circadian clock, determines whether AP-1 drives a transient, protective inflammatory response or a sustained, senescence‑promoting signal. Restoring the proper phase relationship between JNK activity peaks and circadian chromatin accessibility prevents the shift from acute inflammation to chronic inflammaging while preserving host defense.

Mechanistic Basis

The circadian BMAL1:CLOCK complex rhythmically opens chromatin at AP‑1‑containing enhancers (measured by H3K27ac) in macrophages, creating a narrow window of transcriptional competence [1]. JNK phosphorylates c‑Jun to enable AP‑1 DNA binding [2]. We propose that a circadian‑regulated dual‑specificity phosphatase (e.g., DUSP1) is expressed in anti‑phase to JNK activity, generating a pulsatile phosphorylation‑dephosphorylation cycle. When BMAL1 loss desynchronizes chromatin opening, JNK activity persists outside the permissive window, leading to:

• Prolonged c‑Jun phosphorylation at non‑canonical sites
• Increased phosphorylation of downstream targets such as p53 and ATF2 that drive SASP expression
• Mitochondrial ROS‑mediated feed‑forward activation of JNK [3] Thus, the clock does not merely gate timing; it enforces a biochemical threshold that converts JNK from a transient effector into a chronic stress signal when misaligned.

Testable Predictions

1. In WT macrophages, phospho‑JNK (Thr183/Tyr185) exhibits a ~6‑hour peak aligned with maximal H3K27ac at AP‑1 enhancers; Bmal1‑/‑ cells show flattened, elevated phospho‑JNK across 24 h.
2. Pharmacological inhibition of DUSP1 will lengthen the phospho‑JNK pulse and increase senescence markers (p16^INK4a, SA‑β‑gal) even under intact circadian conditions.
3. Timed administration of a JNK inhibitor restricted to the endogenous phospho‑JNK trough will reduce SASP without compromising acute TNF‑induced cytokine production.
4. Rescue of BMAL1 expression via inducible, circadian‑phase‑specific transduction restores phospho‑JNK rhythmicity and lowers inflammaging markers in aged mice.

Experimental Design

• Time‑course phosphoproteomics: Harvest bone‑marrow‑derived macrophages from WT and Bmal1‑/‑ mice every 2 h for 24 h after LPS/TNF stimulation; quantify phospho‑JNK and global phospho‑sites via SILAC‑MS.
• ChIP‑seq & CUT&Tag: Map H3K27ac and phospho‑c‑Jun occupancy at AP‑1 sites across the same time points to assess phase alignment.
• DUSP1 modulation: Use CRISPRi to knock down DUSP1 or overexpress a phosphatase‑dead mutant; measure phospho‑JNK duration and senescence readouts.
• Chronopharmacology: Administer JNK inhibitor (SP600125) at predicted peak vs trough times in vivo (aged WT mice) and evaluate circulating IL‑6, tissue p16^INK4a, and frailty index.
• Phase‑specific BMAL1 rescue: Deliver AAV‑BMAL1 under a Rev‑erbα promoter (active at circadian trough) to Bmal1‑/‑ macrophages; assess restoration of JNK rhythm and inflammaging.

Falsification would occur if phospho‑JNK shows no circadian variation, if DUSP1 manipulation does not alter JNK pulse width, or if timed JNK inhibition fails to dissociate acute cytokine production from chronic SASP induction. This framework directly tests whether the circadian clock’s primary anti‑aging role lies in enforcing the temporal precision of stress‑kinase signaling rather than merely modulating its amplitude.