Background: Epigenetic remodeling is a hallmark of aging hematopoietic stem and progenitor cells (HSPCs). Loss of DNA methylation and heterochromatin dysfunction are two key epigenetic signatures in aged HSPCs, contributing to functional decline and age-associated clonal hematopoiesis (CH). CH promotes the expansion of mutant clones, increasing the risk for hematologic malignancies and other age-related diseases such as cardiovascular disease. Our previous work demonstrated that age-related DNA methylation loss in aged HSPCs significantly disrupts the nuclear organization of H3K9me3-marked heterochromatin and contributes to functional decline, yet the causal mechanisms remain unclear.

Methods: To investigate these mechanisms, we employed an in vitro replication-dependent senescence model. This approach is based on previous findings showing that during cellular replication, human primary fibroblasts form senescence-associated heterochromatin foci (SAHF), characterized by the accumulation of H3K9me3-marked heterochromatin into distinct, bright punctate foci surrounded by H3K27me3 within the nucleus, mimicking the chromatin architecture observed in aged HSPCs. To investigate the causal relationship between DNA methylation and heterochromatin dysfunction in senescence and aging, we perturbed DNA methylation by inhibiting DNMT1 in early-passage cells or overexpressing DNMT1 in late-passage cells. SAHF formation and senescence-associated phenotypes were assessed. DNA methylation was profiled via whole-genome bisulfite sequencing (WGBS), and the genomic localization of H3K9me3 and H3K27me3 was evaluated using CUT&Tag.

Results: DNMT1 expression significantly declined during replication, confirmed by qPCR and western blot analyses comparing early and late passages. Correspondingly, global DNA methylation levels were reduced in replication-induced senescent cells, as measured by dot-blot analysis. To further investigate whether DNMT1 loss accelerates senescence, early-passage cells were treated with a DNMT1 inhibitor. We observed a dramatic increase in senescence-associated β-galactosidase (SA-β-gal) activity, suggesting that loss of DNMT1 accelerates senescence. Conversely, DNMT1 overexpression in late-passage cells (via lentiviral transduction) restored replication capacity and significantly reduced SA-β-gal activity compared to controls, underscoring a critical role for DNMT1 in regulating cellular senescence. Immunofluorescence staining revealed a strong positive correlation between DNMT1 loss and SAHF formation, with increased internal H3K9me3 and surrounding H3K27me3 foci. Notably, young Dnmt1-deficient HSPCs displayed similar chromatin reorganization and H3K9me3 relocalization, alongside impaired colony-forming capacity and disrupted perinuclear heterochromatin, mirroring features of aged HSPCs. Restoration of DNMT1 in late-passage cells markedly reduced SAHF formation. Epigenomic profiling via WGBS and CUT&Tag demonstrated that regions with reduced DNA methylation exhibited extensive redistribution of H3K9me3 and H3K27me3, particularly at genes associated with aging, inflammation, and stem cell function.

Conclusion: Together, these findings demonstrate the essential role of DNMT1-mediated DNA methylation in maintaining heterochromatin integrity. Age-related DNA methylation loss profoundly impacts heterochromatin organization and genome architecture, driving cellular functional decline. Critically, loss of DNA methylation is sufficient to induce SAHF formation in somatic cells and chromatin remodeling in HSPCs, linking epigenetic erosion to stem cell dysfunction. This work provides a mechanistic foundation connecting epigenetic erosion and cellular aging and highlights the potential for therapeutic targeting of epigenetic regulators to reverse senescence and age-related hematopoietic decline.

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2025
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