The PRC2 DNA-methylation substrate of aging is a proliferation-linked mitotic clock, not an organ-margin substrate: a pre-registered cross-organ test of the Organ-Bifurcation hypothesis

Partial epigenetic reprogramming reverses molecular hallmarks of aging, but its therapeutic window — the exposure gap between rejuvenation and neoplastic/identity-loss risk — is thought to be organ-specific. A recently proposed decision model, the Organ-Bifurcation hypothesis, holds that this margin is set by two pre-treatment tissue properties (proliferative index and checkpoint/PRC2 reserve) and that the reversible chromatin substrate on which rejuvenation acts is concentrated in wide-margin, post-mitotic organs (e.g., retina, heart, neurons). We pre-registered and executed a cross-organ test of the substrate premise of this hypothesis at the DNA-methylation level. Using genome-wide methylation (reduced-representation and whole-genome bisulfite sequencing) from eight mouse organs across two independent datasets, we quantified the enrichment of age-associated methylation change at Polycomb Repressive Complex 2 (PRC2) targets (Λ), and — using paired young/old/partially-reprogrammed skin — decomposed it into a reprogramming-reversible component (Λ_rev) and a non-reversible/mitotic component (Λ_mit). Contrary to the hypothesis, both components increase with tissue proliferation and are near-absent in post-mitotic organs: Λ_rev versus proliferative index Spearman ρ = +0.85 (exact-permutation p = 0.006; leave-one-organ-out ρ = +0.77 to +0.93; every proliferative organ exceeded every post-mitotic organ). The reprogramming-reversible substrate — the very target of rejuvenation — is therefore concentrated where the hypothesis predicts the margin is narrowest. Partial reprogramming reversed ~45% of PRC2 age-hypermethylation in skin (H3 supported); H3K27me3 was strongly enriched at PRC2 promoters (3.9× over background), confirming the substrate identity, with a small age-associated loss that is directionally consistent with published data but not statistically resolvable at this resolution (n=2/3). A human multi-tissue test was platform-limited: the only available human multi-organ dataset uses a curated array that demonstrably under-samples the signal — the mouse correlation also collapses on those same loci (ρ 0.85→0.34) — so the human null does not overturn the mouse result, though a residual species/composition contribution cannot be excluded and cross-species generalization remains open. We conclude that the rejuvenation–transformation margin is not written in the PRC2 DNA-methylation substrate at genome-wide resolution in mouse; the substrate is a mitotic clock. This exposes the study's sharpest open question, which we frame as an implication to be tested directly rather than a result: in the post-mitotic organs where partial reprogramming is safest and functionally effective (retina, brain, heart), there is almost no PRC2 promoter methylation to reverse — so what, if not PRC2 DNA methylation, is the reversible substrate of rejuvenation in those tissues?