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Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

Hänzelmann S, Beier F, Gusmao EG, Koch CM, Hummel S, Charapitsa I, Joussen S, Benes V, Brümmendorf TH, Reid G, Costa IG, Wagner W - Clin Epigenetics (2015)

Bottom Line: DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages.Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Centre for Clinical Research (IZKF), RWTH University Medical School, Aachen, Germany ; Institute for Biomedical Technology - Cell Biology, RWTH University Medical School, Aachen, Germany.

ABSTRACT

Background: Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome.

Results: In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.

Conclusions: Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

No MeSH data available.


Related in: MedlinePlus

Telomere distribution in senescent fibroblasts. Telomeres were analyzed by Q-Fish (labeled with Cy3) in nuclei of fibroblasts of early or late passage (n = 3). The nuclear region was counterstained with DAPI. An overview of a cytospin (A) and enlarged nuclei at early and late passage (B) are exemplarily depicted. Separation of nuclear zones in the border, middle, and center is indicated by yellow, white, and violet lines, respectively (size bar = 5 μm). Overall, the nuclear area was greatly increased in cells of late passage (C) and nuclei became more elongated (D). Telomere length markedly decreased in fibroblasts of late passage (E) (a.u. = arbitrary units; error bars depict standard error of nuclei analyzed; early passage: 374 nuclei; late passage: 151 nuclei). The distribution of telomeres changed upon senescence: in early passages (purple dots), they were primarily localized in border and middle regions, whereas distribution changed towards the nuclear center in late passages (yellow dots, data from three biological replica, t-test in all statistical analyses) (F).
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Fig1: Telomere distribution in senescent fibroblasts. Telomeres were analyzed by Q-Fish (labeled with Cy3) in nuclei of fibroblasts of early or late passage (n = 3). The nuclear region was counterstained with DAPI. An overview of a cytospin (A) and enlarged nuclei at early and late passage (B) are exemplarily depicted. Separation of nuclear zones in the border, middle, and center is indicated by yellow, white, and violet lines, respectively (size bar = 5 μm). Overall, the nuclear area was greatly increased in cells of late passage (C) and nuclei became more elongated (D). Telomere length markedly decreased in fibroblasts of late passage (E) (a.u. = arbitrary units; error bars depict standard error of nuclei analyzed; early passage: 374 nuclei; late passage: 151 nuclei). The distribution of telomeres changed upon senescence: in early passages (purple dots), they were primarily localized in border and middle regions, whereas distribution changed towards the nuclear center in late passages (yellow dots, data from three biological replica, t-test in all statistical analyses) (F).

Mentions: Nuclei and telomeres were analyzed in human fibroblasts at early (P3 to P5) and corresponding late passages (P21 to P40) with regard to nuclear area and by quantitative fluorescent in situ hybridization (Q-Fish) with telomere repeat probes (Figure 1A,B). Overall, nuclear area, as defined by optical sections, increased significantly during culture expansion (P < 0.0001; t-test; Figure 1C), whereas nuclear thickness remained relatively constant (5 to 7 μm in z-stacks). Furthermore, nuclei acquired an elongated morphology (Figure 1D). As anticipated, telomere length decreased at later passages (P < 0.0001; Figure 1E). Localization of telomeres within the nucleus was segmented into either the peripheral region, middle region, or central region [23]. In early passages, telomeres were predominantly localized at border regions close to the nuclear lamina while they appeared to be redistributed to the nuclear center at later passages (Figure 1F). Changes in nuclear size, morphology, and localization of telomeres reflect chromosomal reorganization during in vitro culture expansion.Figure 1


Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

Hänzelmann S, Beier F, Gusmao EG, Koch CM, Hummel S, Charapitsa I, Joussen S, Benes V, Brümmendorf TH, Reid G, Costa IG, Wagner W - Clin Epigenetics (2015)

Telomere distribution in senescent fibroblasts. Telomeres were analyzed by Q-Fish (labeled with Cy3) in nuclei of fibroblasts of early or late passage (n = 3). The nuclear region was counterstained with DAPI. An overview of a cytospin (A) and enlarged nuclei at early and late passage (B) are exemplarily depicted. Separation of nuclear zones in the border, middle, and center is indicated by yellow, white, and violet lines, respectively (size bar = 5 μm). Overall, the nuclear area was greatly increased in cells of late passage (C) and nuclei became more elongated (D). Telomere length markedly decreased in fibroblasts of late passage (E) (a.u. = arbitrary units; error bars depict standard error of nuclei analyzed; early passage: 374 nuclei; late passage: 151 nuclei). The distribution of telomeres changed upon senescence: in early passages (purple dots), they were primarily localized in border and middle regions, whereas distribution changed towards the nuclear center in late passages (yellow dots, data from three biological replica, t-test in all statistical analyses) (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356053&req=5

Fig1: Telomere distribution in senescent fibroblasts. Telomeres were analyzed by Q-Fish (labeled with Cy3) in nuclei of fibroblasts of early or late passage (n = 3). The nuclear region was counterstained with DAPI. An overview of a cytospin (A) and enlarged nuclei at early and late passage (B) are exemplarily depicted. Separation of nuclear zones in the border, middle, and center is indicated by yellow, white, and violet lines, respectively (size bar = 5 μm). Overall, the nuclear area was greatly increased in cells of late passage (C) and nuclei became more elongated (D). Telomere length markedly decreased in fibroblasts of late passage (E) (a.u. = arbitrary units; error bars depict standard error of nuclei analyzed; early passage: 374 nuclei; late passage: 151 nuclei). The distribution of telomeres changed upon senescence: in early passages (purple dots), they were primarily localized in border and middle regions, whereas distribution changed towards the nuclear center in late passages (yellow dots, data from three biological replica, t-test in all statistical analyses) (F).
Mentions: Nuclei and telomeres were analyzed in human fibroblasts at early (P3 to P5) and corresponding late passages (P21 to P40) with regard to nuclear area and by quantitative fluorescent in situ hybridization (Q-Fish) with telomere repeat probes (Figure 1A,B). Overall, nuclear area, as defined by optical sections, increased significantly during culture expansion (P < 0.0001; t-test; Figure 1C), whereas nuclear thickness remained relatively constant (5 to 7 μm in z-stacks). Furthermore, nuclei acquired an elongated morphology (Figure 1D). As anticipated, telomere length decreased at later passages (P < 0.0001; Figure 1E). Localization of telomeres within the nucleus was segmented into either the peripheral region, middle region, or central region [23]. In early passages, telomeres were predominantly localized at border regions close to the nuclear lamina while they appeared to be redistributed to the nuclear center at later passages (Figure 1F). Changes in nuclear size, morphology, and localization of telomeres reflect chromosomal reorganization during in vitro culture expansion.Figure 1

Bottom Line: DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages.Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Centre for Clinical Research (IZKF), RWTH University Medical School, Aachen, Germany ; Institute for Biomedical Technology - Cell Biology, RWTH University Medical School, Aachen, Germany.

ABSTRACT

Background: Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome.

Results: In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.

Conclusions: Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

No MeSH data available.


Related in: MedlinePlus