Aging muscle stem cells (MuSCs) exhibit a two‑phase epigenomic decline: an early inflammatory surge followed by late‑stage super‑enhancer redistribution and 3D genome disorganization [1]. We hypothesize that persistent NFκB activation in the inflammatory phase directly triggers the formation of transcriptional condensates via liquid‑liquid phase separation (LLPS), which ectopicly recruits BRD4, Mediator, and H3K27ac‑writing complexes to distal genomic sites. These NFκB‑driven condensates act as neonucleating super‑enhancers that rewire enhancer‑promoter contacts, erode TAD boundaries, and ultimately lock MuSCs into a lineage‑drifted state.

Mechanistically, NFκB p65 contains intrinsically disordered regions that promote LLPS when phosphorylated by IKKβ. In aged MuSCs, chronic IFN‑γ/TNFα signaling sustains p65 phosphorylation, lowering the saturation concentration for condensate formation. These condensates preferentially associate with loci enriched for NFκB motifs and retrotransposable elements, explaining the observed sex‑specific upregulation of retroviral elements in geriatric males [1]. By sequestering co‑activators away from canonical myogenic promoters (e.g., Myf5, Myf6), NFκB condensates reduce promoter‑enhancer looping despite unchanged promoter activity, accounting for the collagen downregulation paradox.

To test this, we will generate MuSC‑specific IKKβ conditional knockout (IKKβ^fl/fl; Pax7‑CreER) mice and compare them to littermate controls across young, old, and geriatric ages. First, we will perform CUT&RUN for H3K27ac and BRD4 to map super‑enhancer landscapes; we predict that IKKβ loss will prevent the shift of super‑enhancers from promoter‑proximal to distal intergenic/intronic regions in old and geriatric MuSCs. Second, we will conduct high‑resolution Hi-C (≈1 billion reads) to quantify TAD boundary insulation and enhancer‑promoter contacts; we expect IKKβ deletion to preserve boundary strength (>30% higher insulation) and maintain Myf5/Myf6 loop frequency. Third, functional assays—single‑fiber transplantation and in vivo injury regeneration—will assess whether preventing super‑enhancer rewiring rescues MuSC proliferative capacity and myogenic output.

A falsifiable outcome is that IKKβ deletion fails to alter super‑enhancer positioning or TAD architecture despite reducing NFκB target gene expression. In that case, the hypothesis would be refuted, suggesting that inflammatory signaling acts upstream but not directly through LLPS‑mediated enhancer rewiring. Conversely, confirmation would establish a causal mechanistic link between early inflammation, phase‑separated enhancer nucleation, and late‑stage 3D genome collapse, offering a concrete target (IKKβ or p65 LLPS inhibitors) to delay or reverse stem cell aging.