Hypothesis

Core idea: Age‑dependent activation of Cdc42 disrupts LaminA/C‑mediated lamina‑associated domains (LADs), which in turn destabilizes HP1α liquid‑liquid phase separation at pericentromeric heterochromatin. This loss of HP1α condensates permits ectopic binding of pioneer transcription factors to distal enhancers, driving the increased accessibility and transcriptional noise observed in aged MuSCs and HSCs.

Mechanistic steps

1. In young stem cells, active Cdc42 maintains a balanced pool of GTP‑bound Cdc42 that supports LaminA/C phosphorylation and proper tethering of chromatin to the nuclear periphery.
2. With ageing, chronic oxidative stress elevates Cdc42‑GTP levels, leading to hyperphosphorylation of LaminA/C, weakening its interaction with LADs and causing chromosome 11 (and other LAD‑rich) territories to detach from the lamina.
3. Detached LADs lose the compact environment that promotes HP1α phase separation; HP1α becomes diffusely nuclear, reducing its capacity to form heterochromatic droplets that sequester enhancer‑like sequences.
4. The consequent de‑compaction exposes distal regulatory motifs (e.g., MEF2, Pax7 sites) to nucleosome remodeling complexes and pioneer factors such as FOXO1, increasing ATAC‑seq signal at those sites.
5. The resulting enhancer noise blunts asymmetric division outcomes: daughter cells receive similar, noisy transcriptional programs, erasing the epigenetic asymmetry that underlies stem‑cell self‑renewal.

Testable predictions

• Prediction 1: Pharmacologic Cdc42 inhibition (e.g., CASIN) in aged MuSCs will restore LaminA/C‑LAD interactions, visualized by DamID or lamin‑ChIP‑seq, and increase the fraction of chromatin attached to the lamina.
• Prediction 2: The same treatment will revive HP1α droplet formation, detectable by fluorescence recovery after photobleaching (FRAP) showing reduced mobility and increased condensate size in aged MuSCs.
• Prediction 3: Restored HP1α phase separation will correlate with a selective decrease in ATAC‑seq peaks at distal enhancers near lineage‑defining genes (Pax7, MyoD) without affecting promoter accessibility.
• Prediction 4: Single‑cell ATAC‑seq combined with lineage tracing will show re‑establishment of asymmetric accessibility patterns between sister MuSCs after Cdc42 inhibition, mirroring the patterns seen in young cells.
• Prediction 5: Expressing a phospho‑deficient LaminA/C mutant (LaminA/C S22A/S390A) in aged MuSCs will phenocopy Cdc42 inhibition, rescuing HP1α condensates and reducing enhancer noise, whereas a phospho‑mimetic mutant will block the rescue.

Experimental approach

• Isolate MuSCs from young (3 mo) and aged (24 mo) mice; treat aged cells with CASIN or vehicle for 48 h.
• Perform DamID‑seq for LaminA/C to quantify LAD‑lamina contacts.
• Conduct immunofluorescence for HP1α and FRAP analysis to assess phase‑separation dynamics.
• Execute scATAC‑seq (10x) on treated and untreated aged MuSCs, focusing on distal enhancer peaks near Pax7, MyoD, and other myogenic regulators.
• Use Cre‑based lineage‑tracing (Pax7‑CreER;Rosa26‑tdTomato) to follow sister cell divisions and assay accessibility asymmetry via scATAC‑seq of isolated sister pairs.
• Complement with rescue experiments using lentiviral expression of LaminA/C mutants.

Falsifiability If Cdc42 inhibition fails to restore LaminA/C‑LAD contacts, HP1α phase separation, or the asymmetric accessibility pattern, or if the phospho‑deficient LaminA/C does not phenocopy the drug effect, the hypothesis would be refuted. Conversely, confirmation of these steps would support a mechanistic link between Cdc42 signaling, nuclear lamina integrity, heterochromatin phase separation, and age‑related chromatin accessibility erosion.

Broader implication This model posits that the nuclear lamina acts as a scaffold for heterochromatin phase separation, and that its age‑dependent destabilization via Cdc42 is a upstream driver of the enhancer accessibility erosion seen across stem cell types. Targeting this axis could therefore provide a unified strategy to rejuvenate chromatin architecture in diverse aged stem cell populations.