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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

Transcription factor binding sites in differentially methylated regions. Analysis of transcription factor binding sites was performed in DMRs and promoter regions of up-/downregulated genes. The heatmap shows the -log10 P value for motifs enriched in at least one DMR signature (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (A). Motifs for the five most significant transcription factors are depicted (B). Subsequently, binding sites in promoter regions of either up- or downregulated genes were analyzed, and motifs common in both lists are marked in bold (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (C).
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Fig5: Transcription factor binding sites in differentially methylated regions. Analysis of transcription factor binding sites was performed in DMRs and promoter regions of up-/downregulated genes. The heatmap shows the -log10 P value for motifs enriched in at least one DMR signature (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (A). Motifs for the five most significant transcription factors are depicted (B). Subsequently, binding sites in promoter regions of either up- or downregulated genes were analyzed, and motifs common in both lists are marked in bold (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (C).

Mentions: Next, we performed a transcription factor (TF) binding site analysis in regions with SA-DNAm changes (450k BeadChip and MethylCap-seq data): 51 motifs were significantly enriched (P value < 0.05; Fisher’s Exact test) in senescence-associated DMRs of at least one fibroblast sample or of MSCs. Most of these TF binding motifs were significantly enriched in both hypermethylated and hypomethylated regions (Figure 5A). Significantly overrepresented motifs include binding sites for early growth response protein 1 (EGR1), activating enhancer-binding protein 2 (TFAP2A), protein C-ets-1 (ETS1), neuroblastoma MYC oncogene (MYCN), and aryl hydrocarbon receptor nuclear translocator (ARNT; Figure 5B). Enrichment of these TF binding sites suggests that they are either directly involved in the regulation of SA-DNAm changes or that their binding is influenced by differential methylation and hence relevant for gene expression changes.Figure 5


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)

Transcription factor binding sites in differentially methylated regions. Analysis of transcription factor binding sites was performed in DMRs and promoter regions of up-/downregulated genes. The heatmap shows the -log10 P value for motifs enriched in at least one DMR signature (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (A). Motifs for the five most significant transcription factors are depicted (B). Subsequently, binding sites in promoter regions of either up- or downregulated genes were analyzed, and motifs common in both lists are marked in bold (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Transcription factor binding sites in differentially methylated regions. Analysis of transcription factor binding sites was performed in DMRs and promoter regions of up-/downregulated genes. The heatmap shows the -log10 P value for motifs enriched in at least one DMR signature (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (A). Motifs for the five most significant transcription factors are depicted (B). Subsequently, binding sites in promoter regions of either up- or downregulated genes were analyzed, and motifs common in both lists are marked in bold (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction, adj. P value < 0.05) (C).
Mentions: Next, we performed a transcription factor (TF) binding site analysis in regions with SA-DNAm changes (450k BeadChip and MethylCap-seq data): 51 motifs were significantly enriched (P value < 0.05; Fisher’s Exact test) in senescence-associated DMRs of at least one fibroblast sample or of MSCs. Most of these TF binding motifs were significantly enriched in both hypermethylated and hypomethylated regions (Figure 5A). Significantly overrepresented motifs include binding sites for early growth response protein 1 (EGR1), activating enhancer-binding protein 2 (TFAP2A), protein C-ets-1 (ETS1), neuroblastoma MYC oncogene (MYCN), and aryl hydrocarbon receptor nuclear translocator (ARNT; Figure 5B). Enrichment of these TF binding sites suggests that they are either directly involved in the regulation of SA-DNAm changes or that their binding is influenced by differential methylation and hence relevant for gene expression changes.Figure 5

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