DNA Methylation Changes Upon Senescence are Strand-Specific and Reflect Chromatin Conformation

Abstract

Replicative senescence of cells is associated with reproducible DNA methylation (DNAm) changes at specific sites in the genome. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated upon culture expansion of mesenchymal stem cells and other cell types. During reprogramming into induced pluripotent stem cells, 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. 4C analysis of senescence-associated CpGs revealed reproducible interaction changes during senescence without evidence for preferential interaction between senescence-associated CpGs. These results indicate that senescence-associated DNAm is not regulated in a targeted manner but rather caused by higher order chromatin conformation states.

Keywords: Cellular Aging, DNA methylation, epigenetic, hairpin, hemimethylation, mesenchymal stem cells, senescence, 5mC, 4C