Youthful circulation reshapes age-associated DNA methylation across blood and liver.

Abstract

Prolonged heterochronic parabiosis (HPB) reduces DNA methylation (DNAm) age in old mice, but how age-opposing methylation changes are organized across tissues and retained after removal of youthful circulation is unclear. We analyzed processed reduced-representation bisulfite sequencing data from 78 blood and liver samples from a published prolonged-HPB experiment. Across 1,008,133 CpGs with at least 10-fold coverage in every sample, we quantified normal-aging and HPB effects, defined directional reversal by effect size, tested exact cross-tissue and post-detachment persistence, and used chromosome-aware null models for overlap and gene-convergence analyses. HPB-associated methylation changes opposed normal aging in blood and liver, including in independent-cohort comparisons that avoided a shared Old-ISO reference. At the primary 10-percentage-point threshold, 22,424 blood and 22,286 liver CpGs were reversed immediately after HPB; 1,792 were shared at identical loci. After two months of recovery, 25,867 blood and 15,437 liver CpGs were reversed, with 1,387 exact shared loci. A 64-CpG cross-tissue core persisted across both time points. Reversed CpGs were depleted from promoters and CpG islands and enriched in gene bodies and open-sea regions. After exact shared CpGs were removed, tissue-specific sites showed a modest excess of nearest-gene convergence (4,716 observed vs 4,215 expected; empirical P≈.001), whereas promoter/gene-body and Gene Ontology convergence were not supported by stricter genomic null models. Youthful circulation produces both conserved and tissue-specific reversal of age-associated methylation. Persistence after detachment and modest cross-tissue genomic-neighborhood convergence suggest that systemic rejuvenation is encoded at multiple levels of epigenomic organization.