Accelerated methylation of ribosomal RNA genes during the cellular senescence of Werner syndrome fibroblasts

Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organis mal aging. The role of rDNA in premature and normal human aging was investi gated by measuring rDNA gene copy number, the level of rDNA methylation, an d rRNA expression during the in vitro senescence of primary fibroblasts fro m normal (young and old) donors and from Werner syndrome (WS) patients, In comparison to their normal counterparts, WS fibroblasts grew slowly and rea ched senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts, H owever, in senescent WS and normal old fibroblasts, we detected rDNA specie s with unusually slow electrophoretic mobility, Cellular aging in Saccharom yces cerevisiae is accompanied by the formation and accumulation of rDNA ci rcles. Our analysis revealed that the rDNA species observed in this study w ere longer, linear rDNA molecules attributable to the inhibition of EcoRI c leavage by methylation. Furthermore, isoschizomeric restriction analysis co nfirmed that in vitro senescence of fibroblasts is accompanied by significa nt increases in cytosine methylation within rDNA genes, This increased meth ylation is maximal during the abbreviated life span of WS fibroblasts. Desp ite increased methylation of rDNA in senescent cells, the steady-state leve ls of 28S rRNA remained constant over the life span of both normal and WS f ibroblasts.