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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

Senescence-associated hypomethylation is enriched in lamina-associated domains. DNAm profiles of fibroblasts at early and late passage (MethylCap-seq) were compared to previously published data on H3K27me3 [30], H3K4me3 [30], H3K4me1 [30], H3K9me3 [30], and lamina-associated domains (LADs) [29] in fibroblasts. Non-methylated DNA was particularly associated with the histone mark H3K9me3 and LADs, whereas H3K27me3, H3K4me3, and H3K4me1 were significantly reduced in these regions. RPKM signals are exemplarily depicted for a region in chromosome 16 (A). Distributions of average RPKM levels of H3K27me3 (B), H3K4me3 (C), H3K4me1 (D), and H3K9me3 (E) in 1,000-bp windows around DMRs are shown. Average signal intensity of DNAm was significantly lower inside LADs than outside LADs (Mann-Whitney test of equal means) (F). A particular sharp decline of DNAm level was observed at the border of LADs (in all samples) (G). Senescence-associated DMRs were then correlated with LADs. The proportion of senescence-associated (SA) hypermethylation was significantly decreased in LADs while SA-hypomethylation was highly significantly increased in LADs as compared to randomly selected regions (two-tailed Fisher’s Exact test) (H).
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Fig3: Senescence-associated hypomethylation is enriched in lamina-associated domains. DNAm profiles of fibroblasts at early and late passage (MethylCap-seq) were compared to previously published data on H3K27me3 [30], H3K4me3 [30], H3K4me1 [30], H3K9me3 [30], and lamina-associated domains (LADs) [29] in fibroblasts. Non-methylated DNA was particularly associated with the histone mark H3K9me3 and LADs, whereas H3K27me3, H3K4me3, and H3K4me1 were significantly reduced in these regions. RPKM signals are exemplarily depicted for a region in chromosome 16 (A). Distributions of average RPKM levels of H3K27me3 (B), H3K4me3 (C), H3K4me1 (D), and H3K9me3 (E) in 1,000-bp windows around DMRs are shown. Average signal intensity of DNAm was significantly lower inside LADs than outside LADs (Mann-Whitney test of equal means) (F). A particular sharp decline of DNAm level was observed at the border of LADs (in all samples) (G). Senescence-associated DMRs were then correlated with LADs. The proportion of senescence-associated (SA) hypermethylation was significantly decreased in LADs while SA-hypomethylation was highly significantly increased in LADs as compared to randomly selected regions (two-tailed Fisher’s Exact test) (H).

Mentions: We then compared DNAm changes resulting from long-term culture with previously published datasets on posttranslational histone modifications [25] and on lamina-associated domains (LADs) [26] in human fibroblasts (Figure 3A). Genomic regions with SA-hypermethylation in late-passage samples from fibroblasts and MSCs revealed significant enrichment in regions with trimethylation on histone 3 at lysine 27 (H3K27me3; not observed in fibro 1), trimethylation on histone 3 at lysine 4 (H3K4me3), and monomethylation on histone 3 at lysine 4 (H3K4me1) (Figure 3B,C,D). H3K27me3 is characteristic for inactivated chromatin within gene-rich regions, while H3K4me3 and H3K4me1 are, respectively, indicative of active promoters and enhancers [27]. In contrast, H3K9me3, a repressive histone mark mainly occurring in gene-poor regions, was associated with non-methylated regions. All samples analyzed had a significant presence of SA-hypomethylation in genomic regions with H3K9me3 marks (Figure 3E). Thus, specific histone modifications are enriched in regions with DNAm changes during long-term culture.Figure 3


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)

Senescence-associated hypomethylation is enriched in lamina-associated domains. DNAm profiles of fibroblasts at early and late passage (MethylCap-seq) were compared to previously published data on H3K27me3 [30], H3K4me3 [30], H3K4me1 [30], H3K9me3 [30], and lamina-associated domains (LADs) [29] in fibroblasts. Non-methylated DNA was particularly associated with the histone mark H3K9me3 and LADs, whereas H3K27me3, H3K4me3, and H3K4me1 were significantly reduced in these regions. RPKM signals are exemplarily depicted for a region in chromosome 16 (A). Distributions of average RPKM levels of H3K27me3 (B), H3K4me3 (C), H3K4me1 (D), and H3K9me3 (E) in 1,000-bp windows around DMRs are shown. Average signal intensity of DNAm was significantly lower inside LADs than outside LADs (Mann-Whitney test of equal means) (F). A particular sharp decline of DNAm level was observed at the border of LADs (in all samples) (G). Senescence-associated DMRs were then correlated with LADs. The proportion of senescence-associated (SA) hypermethylation was significantly decreased in LADs while SA-hypomethylation was highly significantly increased in LADs as compared to randomly selected regions (two-tailed Fisher’s Exact test) (H).
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Related In: Results  -  Collection

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Fig3: Senescence-associated hypomethylation is enriched in lamina-associated domains. DNAm profiles of fibroblasts at early and late passage (MethylCap-seq) were compared to previously published data on H3K27me3 [30], H3K4me3 [30], H3K4me1 [30], H3K9me3 [30], and lamina-associated domains (LADs) [29] in fibroblasts. Non-methylated DNA was particularly associated with the histone mark H3K9me3 and LADs, whereas H3K27me3, H3K4me3, and H3K4me1 were significantly reduced in these regions. RPKM signals are exemplarily depicted for a region in chromosome 16 (A). Distributions of average RPKM levels of H3K27me3 (B), H3K4me3 (C), H3K4me1 (D), and H3K9me3 (E) in 1,000-bp windows around DMRs are shown. Average signal intensity of DNAm was significantly lower inside LADs than outside LADs (Mann-Whitney test of equal means) (F). A particular sharp decline of DNAm level was observed at the border of LADs (in all samples) (G). Senescence-associated DMRs were then correlated with LADs. The proportion of senescence-associated (SA) hypermethylation was significantly decreased in LADs while SA-hypomethylation was highly significantly increased in LADs as compared to randomly selected regions (two-tailed Fisher’s Exact test) (H).
Mentions: We then compared DNAm changes resulting from long-term culture with previously published datasets on posttranslational histone modifications [25] and on lamina-associated domains (LADs) [26] in human fibroblasts (Figure 3A). Genomic regions with SA-hypermethylation in late-passage samples from fibroblasts and MSCs revealed significant enrichment in regions with trimethylation on histone 3 at lysine 27 (H3K27me3; not observed in fibro 1), trimethylation on histone 3 at lysine 4 (H3K4me3), and monomethylation on histone 3 at lysine 4 (H3K4me1) (Figure 3B,C,D). H3K27me3 is characteristic for inactivated chromatin within gene-rich regions, while H3K4me3 and H3K4me1 are, respectively, indicative of active promoters and enhancers [27]. In contrast, H3K9me3, a repressive histone mark mainly occurring in gene-poor regions, was associated with non-methylated regions. All samples analyzed had a significant presence of SA-hypomethylation in genomic regions with H3K9me3 marks (Figure 3E). Thus, specific histone modifications are enriched in regions with DNAm changes during long-term culture.Figure 3

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