From LAD Aging-Program Opposition to a Prospective Architecture-Function Test

This pre-experiment release reports a falsification-first investigation of replicative aging, lamina-associated domain (LAD) methylation, and perturbation-associated opposition to an untreated-aging methylation program. Analysis of the GSE179847 cellular-lifespan methylation dataset identified greater net hypomethylation at independently defined LAD CpGs than at context-matched non-LAD CpGs during untreated replicative aging. Subsequent perturbation analyses identified a reproducible oligomycin-associated methylation response opposing the frozen untreated-aging LAD vector, termed LAD Aging-Program Opposition (LAPO). Importantly, LAPO is not interpreted as rejuvenation. Functional and transcriptomic analyses did not demonstrate corresponding rejuvenation: oligomycin-associated LAPO was dissociable from improved proliferation, reduced cell death, or opposition of the measured SenMayo transcriptomic signature. A sequence of increasingly stringent genomic-specificity analyses (Gates 7G–7I) further showed that the available EPIC-array data cannot identify LAD membership independently of correlated genomic-domain properties, including replication timing, partially methylated domains (PMDs), A/B compartment state, and spatial genomic architecture. Gate 7I reconstructed and validated full-autosomal oligomycin effects across 839,998 probes, demonstrating that missing treatment effects were not the principal limitation; adequate common genomic support remained unavailable under the frozen domain-balancing requirements. These results motivate a prospective experimental program rather than further retrospective claims of LAD specificity. Gate 8 is designed to test the causal chain from perturbation to methylation opposition, physical lamina/chromatin architecture, and functional recovery using direct Lamin-B1 contact measurements, orthogonal radial DNA-FISH, dense methylome profiling, same-system replication timing, PMD and A/B-compartment characterization, and standardized injury-recovery measurements. Gate 8P defines a six-donor technical and biological pilot and freezes key execution, provenance, assay-qualification, injury-dose, comparator, variance, and readiness rules before confirmatory experimentation. No Gate 8 or Gate 8P experimental outcome is reported in this release. No pilot donor has been enrolled or randomized, and no challenge dose or comparator has been qualified. This record documents completed retrospective analyses and prospectively frozen experimental requirements so that future results can be evaluated against decisions established before those results exist. Reserved DOI: 10.5281/zenodo.23113585