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Background: Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome., Results: In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes., Conclusions: Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.