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

Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
The FASEB Journal (Impact Factor: 5.04). 10/2000; 14(12):1715-24.
Source: PubMed


Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.

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    • "Hypermethylation of rDNA promoter was described in brain from Alzheimer’s disease [11] and suicide subjects [12], while methylation levels of 18S and 28S 5’ regions were decreased in white blood cells from systemic lupus erythematosus subjects [13]. rDNA hypermethylation occurs during aging [14], and accordingly accelerated methylation of ribosomal regions was shown in fibroblasts from subjects affected by Werner syndrome [15]. The analysis of rDNA methylation in tumor samples appears to be in this context of extreme interest. "
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    ABSTRACT: Background There is a body of evidence that shows a link between tumorigenesis and ribosome biogenesis. The precursor of mature 18S, 28S and 5.8S ribosomal RNAs is transcribed from the ribosomal DNA gene (rDNA), which exists as 300–400 copies in the human diploid genome. Approximately one half of these copies are epigenetically silenced, but the exact role of epigenetic regulation on ribosome biogenesis is not completely understood. In this study we analyzed the methylation profiles of the rDNA promoter and of the 5’ regions of 18S and 28S in breast cancer. Methods We analyzed rDNA methylation in 68 breast cancer tissues of which the normal counterpart was partially available (45/68 samples) using the MassARRAY EpiTYPER assay, a sensitive and quantitative method with single base resolution. Results We found that rDNA locus tended to be hypermethylated in tumor compared to matched normal breast tissues and that the DNA methylation level of several CpG units within the rDNA locus was associated to nuclear grade and to nucleolar size of tumor tissues. In addition we identified a subgroup of samples in which large nucleoli were associated with very limited or absent rDNA hypermethylation in tumor respect to matched normal tissue. Conclusions In conclusion, we suggest that rDNA is an important target of epigenetic regulation in breast tumors and that rDNA methylation level is associated to nucleolar size.
    Full-text · Article · May 2014 · BMC Cancer
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    • "However, subsequent studies including those in post mortem samples of the human cerebral cortex did not confirm early observations of aging-associated changes in the genomic rDNA content [17] [18]. Likewise, no disruption of rDNA stability has been found in cell culture models of replicative senescence [19] [20]. "
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    ABSTRACT: Homologous recombination (HR)-mediated instability of the repetitively organized ribosomal DNA (rDNA) has been proposed as a mediator of cell senescence in yeast triggering the DNA damage response. High individual variability in the content of human rDNA suggests that this genomic region remained relatively unstable throughout evolution. Therefore, quantitative real time PCR was used to determine the genomic content of rDNA in post mortem samples of parietal cortex from 14 young- and 9 elderly individuals with no diagnosis of a chronic neurodegenerative/neurological disease. In addition, rDNA content in that brain region was compared between 10 age-matched control individuals and 10 patients with dementia with Lewy bodies (DLB) which involves neurodegeneration of the cerebral cortex. Probing rRNA-coding regions of rDNA revealed no effects of aging on the rDNA content. Elevated rDNA content was observed in DLB. Conversely, in the DLB pathology-free cerebellum, lower genomic content of rDNA was present in the DLB group. In the parietal cortex, such a DLB-associated instability of rDNA was not accompanied by any major changes of CpG methylation of the rDNA promoter. As increased cerebro-cortical rDNA content was previously reported in Alzheimer's diseases, neurodegeneration appears to be associated with instability of rDNA. The hypothetical origins and consequences of this phenomenon are discussed including possibilities that the DNA damage-induced recombination destabilizes rDNA and that differential content of rDNA affects heterochromatin formation, gene expression and/or DNA damage response. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.
    Full-text · Article · Jan 2014 · Biochimica et Biophysica Acta
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    • "Nevertheless, long-term changes in the ratio of rRNA genes in an 'open' euchromatic versus a 'closed' heterochromatic structure correlate with cell differentiation, senescence, disease and malignancy . Hypomethylation of rRNA genes, reflecting an increase in the number of active genes at the expense of silent ones, has been associated with uncontrolled growth and rRNA synthesis in several tumors [5] [6], whereas in senescent cells the rDNA promoter was found to be hypermethylated [7] [8]. Given that down-regulation of rRNA synthesis limits ribosome biogenesis and protects cells from energy deprivation-induced apoptosis, elucidation of mechanisms that promote and maintain a fraction of rDNA repeats in a facultative heterochromatic conformation is a key issue that links chromatin silencing to chromosome stability and cell metabolism. "
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    ABSTRACT: rRNA synthesis is regulated by genetic and epigenetic mechanisms. Epigenetic states are metastable, changing in response to appropriate signals, thereby modulating transcription in vivo. The establishment, maintenance and reversal of epigenetic features are fundamental for the cell's ability to 'remember' past events, to adapt to environmental changes or developmental cues and to propagate this information to the progeny. As packaging into chromatin is critical for the stability and integrity of repetitive DNA, keeping a fraction of rRNA genes in a metastable heterochromatic conformation prevents aberrant exchanges between repeats, thus safeguarding nucleolar structure and rDNA stability. In this review, we will focus on the nature of the molecular signatures that characterize a given epigenetic state of rDNA in mammalian cells, including noncoding RNA, DNA methylation and histone modifications, and the mechanisms by which they are established and maintained. This article is part of a Special Issue entitled: Transcription by Odd Pols.
    Full-text · Article · Oct 2012 · Biochimica et Biophysica Acta
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