Hypothesis

Aged muscle satellite cells (MuSCs) lose CTCF/cohesin‑mediated insulation at a subset of topologically associating domain (TAD) boundaries, permitting ectopic contacts between super‑enhancers and endogenous retroviral (ERV) loci. This ERV‑driven interferon signaling feeds back to NFκB, reinforcing further boundary loss in a male‑biased manner due to lower expression of the X‑linked histone demethylase KDM6A, which normally stabilizes CTCF binding.

Mechanistic basis

1. Boundary erosion – Hi‑C data show widespread insulation loss in aged MuSCs, especially at boundaries flanking ERV clusters 1. Reduced CTCF occupancy increases the probability of transient enhancer‑ERV contacts, which, per the transient contact model, can initiate transcription when sustained 2.
2. ERV activation and interferon signaling – Ectopic enhancer contacts trigger ERV transcription, producing double‑stranded RNA that activates cytosolic sensors (e.g., MDA5) and amplifies type‑I interferon responses. Interferon signaling further upregulates NFκB, which phosphorylates CTCF and promotes its eviction from chromatin, creating a positive feedback loop.
3. Sex bias – KDM6A (UTX) demethylates H3K27me3 at CTCF sites, favoring binding. Male MuSCs possess a single X chromosome, resulting in lower basal KDM6A expression than females. Consequently, male cells experience faster boundary erosion under inflammatory stress, explaining the observed male‑specific ERV upregulation in geriatric MuSCs 1.

Predictions

• P1: Acute depletion of CTCF in young MuSCs will reproduce the aged pattern of super‑enhancer redistribution toward ERVs and increase interferon‑stimulated gene expression.
• P2: Overexpression of KDM6A in aged male MuSCs will restore boundary insulation, reduce ERV contacts, and ameliorate inflammatory signaling without altering global H3K27ac levels.
• P3: Female MuSCs will show delayed boundary loss and ERV activation compared with males when exposed to equivalent inflammatory cytokines, unless KDM6A is knocked down.

Experimental design

1. CRISPR‑based boundary editing – Design sgRNAs flanking CTCF sites at TAD boundaries adjacent to ERV‑rich regions in young MuSCs. Generate homozygous deletions and assess (a) Hi‑C insulation scores, (b) H3K27ac ChIP‑seq at super‑enhancers and ERVs, (c) RNA‑seq for ERV transcripts and interferon‑stimulated genes. Compare to aged MuSCs.
2. KDM6A rescue – Transduce aged male MuSCs with a lentiviral KDM6A construct. Perform Cut&Run for CTCF, Hi‑ChIP for H3K27ac‑linked contacts, and measure interferon beta secretion. Evaluate functional outcomes: quiescence fraction (Pax7+/Ki67−), proliferative capacity (EdU incorporation), and myogenic differentiation (MyoHc formation).
3. Sex comparison – Treat young male and female MuSCs with low‑dose TNF‑α to mimic chronic inflammation. Monitor CTCF occupancy over time via CUT&Tag and ERV expression via single‑cell RNA‑seq. Include a KDM6A knockdown arm in females to test sufficiency.

Falsification: If CTCF boundary deletion fails to increase ERV‑associated super‑enhancer activity, or if KDM6A overexpression does not rescue insulation and inflammation in aged male MuSCs, the hypothesis would be refuted. Conversely, confirmation of these predictions would support a mechanistic link between sex‑specific chromatin insulator dynamics, ERV reactivation, and stem‑cell aging.