DNA methylation changes upon replicative senescence of mesenchymal stromal cells

In culture, human mesenchymal stromal cells enter replicative senescence after a limited amount of cell divisions. Thus, they stop proliferation but still stay viable. This phenomenon is not only observed in vitro but also in vivo, where accumulating senescent cells promote aging and tumor progression. This cell cycle exit is accompanied by various changes including cell morphology, gene expression and the epigenetic markup of cells. One of the epigenetic modifications, which is affected by senescence, is the methylation of cytosines at cytosine-guanine dinucleotides (CpG sites). In this thesis, these so-called senescence-associated DNA methylation changes were further characterized. It could be shown, that specific senescence-associated CpG sites are shared between different cell types. Based on these findings, an Epigenetic-Senescence-Signature was established, which predicts passage numbers of multiple cell types by using DNA methylation values at only four CpG sites. Furthermore, we showed that senescence-associated regions exhibit highly dynamic and fluctuating methylation patterns at neighboring CpG sites on single DNA strands. Thus, it seems that CpG sites in direct neighborhood are not synchronously modulated but rather experience methylation or demethylation in a stochastic manner. Consequently, DNA methylation patterns are not suitable to predict clonal composition of mesenchymal stromal cell preparations. The random DNA methylation pattern within senescence-associated regions suggests, that DNA methylation at these sites is not regulated by a protein complex but might rather be induced by the 3D chromatin structure. We could show, that hemimethylation of only one cytosine of the CpG site is frequently and stably maintained at certain senescence-associated CpGs. Hemimethylation has recently been shown to be associated to CCCTC-binding factor (CTCF), which is involved in the looping and three-dimensional conformation of chromatin. Further analysis of the senescence-associated CpG sites by circular chromatin conformation capture revealed reproducible chromatin interaction changes between early and late passages at these sites and confirmed the enrichment of CTCF. Taken together, we observed conserved senescence-associated genomic regions, which either randomly gain or lose DNA methylation at neighboring CpG sites. Therefore, we propose that senescence-associated DNA methylation changes are not modulated in a targeted manner but rather induced by higher order chromatin organization