Abstract
Replicative senescence of cells in culture is associated with highly reproducible DNA methylation (DNAm) changes at specific sites in the genome. Thus far, it is largely unclear if these epigenetic modifications are directly regulated, or if they are randomly evoked by other chromatin changes during long-term culture.
We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypo-methylated in the course of culture expansion of mesenchymal stem cells (MSCs) and other cell types. These modifications provide a biomarker for replicative senescence and correlate with the number of passages in vitro. During reprogramming into induced pluripotent stem cells (iPSCs) senescence-associated DNAm is reversed simultaneously with pluripotency-associated DNAm changes. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that upon passaging the DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development. Notably, BBA-seq of hairpin-linked DNA molecules demonstrated that many CpG dyads are methylated only on the forward or the reverse strand. This hemimethylation was conserved over many passages, indicating that it was not due to insufficient maintenance of DNAm patterns. Circularized chromatin conformation capture (4C) of senescence-associated CpGs revealed reproducible changes during senescence without evidence for preferential interaction between CpGs that become either hyper- or hypomethylated.
Taken together, senescence-associated DNAm fluctuates stochastically at specific sites in the genome. Strand-specific DNAm and reproducible changes in 4C indicate that epigenetic modifications of these CG dyads are not regulated in a targeted manner but rather caused by passage-specific higher order chromatin conformation states.