Hypothesis: Mitochondrial ROS oxidizes the ATPase domain of the nucleosome remodeler CHD4, impairing its sliding activity and leading to progressive loss of accessibility at distal enhancers that sustain stem‑cell identity. This mechanism explains the convergent erosion observed in aged muscle satellite cells, hematopoietic stem cells, and other tissues, and positions CHD4 oxidation as an upstream driver of the AP‑1/NFκB‑signature accessibility gains reported in aging.

In aged stem cells, ROS accumulation damages Fe‑S clusters and cysteine residues in chromatin regulators. Recent work shows that oxidized CHD4 exhibits reduced nucleosome‑sliding velocity in vitro, which would decrease accessibility at enhancers that rely on CHD4‑mediated nucleosome repositioning for TF binding (see CHD4 oxidation studies)[https://doi.org/10.1101/gr.240093.118]. We predict that this loss of remodeling activity precedes the observed decline in ATAC‑seq signal at Pax7 enhancers in muscle satellite cells and at HoxA cluster enhancers in hematopoietic stem cells, thereby causing the downstream loss of stem‑cell identity genes and the compensatory increase in AP‑1/NFκB motif accessibility as nucleosomes become statically positioned and expose cryptic binding sites.

To test this, we will compare young and aged satellite cells and HSCs for (i) CHD4 oxidation state using biotin‑switch assays coupled to mass spectrometry, (ii) CHD4 chromatin‑binding dynamics via FRAP, and (iii) ATAC‑seq signal at defined enhancers. We will then express a redox‑resistant CHD4 mutant (Cys→Ser substitutions in the ATPase domain) via lentiviral transduction in aged cells and assess whether enhancer accessibility, Pax7/HoxA expression, and functional regeneration are restored to youthful levels. Conversely, treating young cells with a mitochondria‑targeted ROS generator (e.g., MitoParaquat) should induce CHD4 oxidation, reduce enhancer accessibility, and phenocopy aging without altering proliferation rates.

If CHD4 oxidation is causal, antioxidant treatment with MitoTEMPO should rescue CHD4 activity and enhancer accessibility, whereas global ROS scavengers that do not target mitochondria will have little effect. Importantly, because CHD4 remodeling does not directly drive cell‑cycle progression, rescuing its activity is unlikely to increase malignant transformation, addressing a key safety concern in epigenetic rejuvenation therapies.

Falsifiable outcomes: (1) No correlation between CHD4 oxidation status and enhancer ATAC‑seq loss across aged tissues; (2) Expression of redox‑resistant CHD4 fails to restore accessibility or regeneration; (3) MitoTEMPO treatment does not improve CHD4 activity or enhancer openness despite lowering bulk ROS. Any of these results would refute the hypothesis and suggest that ROS acts through alternative chromatin modifiers.

The eroding chromatin landscape of aged stem cells provides the foundational observation of progressive accessibility loss. Global chromatin accessibility profiling reveals chronic activation documents the AP‑1/NFκB motif enrichment in aged satellite cells. Distinct causes of hematopoietic aging phenotypes links NFκB dysregulation to myeloid bias. Chromatin accessibility changes in muscle satellite cells identifies the two Pax7 enhancers that lose accessibility with age. Stem cell aging mechanisms and therapeutic opportunities outlines ROS as a key mechanistic driver. Aging alters epigenetic asymmetry of HSC division connects epigenetic asymmetry to functional potential. Remodeling of epigenome and transcriptome with aging notes the widespread induction of inflammatory pathways. The eroding chromatin landscape as therapeutic target highlights the need for reversible, safe interventions.