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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

Gene expression changes upon long-term culture. Gene expression profiles (RNA-seq) were analyzed in MSCs of early passage (P4) and late passage (P13; n = 3). The volcano plot demonstrates differential expression upon long-term culture. Relevant genes are indicated in red (A). Particularly, genes localized at the border of LADs were hardly expressed (B). No significant association of senescence-associated gene expression changes was observed at the border of LADs (C). Differentially expressed genes are in genomic regions that are not localized in LADs (D). Gene ontology (GO) analysis was performed for genes, which were either significantly up- or downregulated compared to all genes (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction). Shown are the most significant categories (E).
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Fig4: Gene expression changes upon long-term culture. Gene expression profiles (RNA-seq) were analyzed in MSCs of early passage (P4) and late passage (P13; n = 3). The volcano plot demonstrates differential expression upon long-term culture. Relevant genes are indicated in red (A). Particularly, genes localized at the border of LADs were hardly expressed (B). No significant association of senescence-associated gene expression changes was observed at the border of LADs (C). Differentially expressed genes are in genomic regions that are not localized in LADs (D). Gene ontology (GO) analysis was performed for genes, which were either significantly up- or downregulated compared to all genes (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction). Shown are the most significant categories (E).

Mentions: To further correlate DNAm changes with gene expression changes, we sequenced the transcriptome of three MSC preparations at early (P3) and late passages (P13; the same MSC preparations previously used for the analysis of DNAm profiles [19]). Six hundred forty-eight genes were downregulated and 499 genes upregulated during long-term culture (FDR < 0.01 and log2 fold change > 2; Figure 4A; Additional file 1: Table S1). Interestingly, among the significantly downregulated genes were lamin B1 (LMNB1; P = 5.9*10 − 13) and lamin B2 (LMNB2; P = 4.1*10−37). Further, downregulated genes included the lamin B receptor (LBR; P = 6.8*10−4), which anchors the lamina and heterochromatin to the membrane; thymopoietin (TMPO; P = 3.8*10−18), which may play a role in the assembly of the nuclear lamina and thus help maintain the structural organization of the nuclear envelope; and spectrin repeat containing nuclear envelope 2 (SYNE2, P = 0.005), whereas SYNE1 was upregulated (P = 3.1*10−18). These results indicate that differential expression of genes involved in the nuclear lamina may contribute to reorganization of chromatin during long-term culture.Figure 4


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)

Gene expression changes upon long-term culture. Gene expression profiles (RNA-seq) were analyzed in MSCs of early passage (P4) and late passage (P13; n = 3). The volcano plot demonstrates differential expression upon long-term culture. Relevant genes are indicated in red (A). Particularly, genes localized at the border of LADs were hardly expressed (B). No significant association of senescence-associated gene expression changes was observed at the border of LADs (C). Differentially expressed genes are in genomic regions that are not localized in LADs (D). Gene ontology (GO) analysis was performed for genes, which were either significantly up- or downregulated compared to all genes (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction). Shown are the most significant categories (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Gene expression changes upon long-term culture. Gene expression profiles (RNA-seq) were analyzed in MSCs of early passage (P4) and late passage (P13; n = 3). The volcano plot demonstrates differential expression upon long-term culture. Relevant genes are indicated in red (A). Particularly, genes localized at the border of LADs were hardly expressed (B). No significant association of senescence-associated gene expression changes was observed at the border of LADs (C). Differentially expressed genes are in genomic regions that are not localized in LADs (D). Gene ontology (GO) analysis was performed for genes, which were either significantly up- or downregulated compared to all genes (Fisher’s Exact test followed by Benjamin Hochberg multiple test correction). Shown are the most significant categories (E).
Mentions: To further correlate DNAm changes with gene expression changes, we sequenced the transcriptome of three MSC preparations at early (P3) and late passages (P13; the same MSC preparations previously used for the analysis of DNAm profiles [19]). Six hundred forty-eight genes were downregulated and 499 genes upregulated during long-term culture (FDR < 0.01 and log2 fold change > 2; Figure 4A; Additional file 1: Table S1). Interestingly, among the significantly downregulated genes were lamin B1 (LMNB1; P = 5.9*10 − 13) and lamin B2 (LMNB2; P = 4.1*10−37). Further, downregulated genes included the lamin B receptor (LBR; P = 6.8*10−4), which anchors the lamina and heterochromatin to the membrane; thymopoietin (TMPO; P = 3.8*10−18), which may play a role in the assembly of the nuclear lamina and thus help maintain the structural organization of the nuclear envelope; and spectrin repeat containing nuclear envelope 2 (SYNE2, P = 0.005), whereas SYNE1 was upregulated (P = 3.1*10−18). These results indicate that differential expression of genes involved in the nuclear lamina may contribute to reorganization of chromatin during long-term culture.Figure 4

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