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. 2013 Dec;15(12):1495-506.
doi: 10.1038/ncb2879. Epub 2013 Nov 24.

Senescent cells harbour features of the cancer epigenome

Hazel A Cruickshanks  1 , Tony McBryan, David M Nelson, Nathan D Vanderkraats, Parisha P Shah, John van Tuyn, Taranjit Singh Rai, Claire Brock, Greg Donahue, Donncha S Dunican, Mark E Drotar, Richard R Meehan, John R Edwards, Shelley L Berger, Peter D Adams
Affiliations

Senescent cells harbour features of the cancer epigenome

Hazel A Cruickshanks et al. Nat Cell Biol. 2013 Dec.

Abstract

Altered DNA methylation and associated destabilization of genome integrity and function is a hallmark of cancer. Replicative senescence is a tumour suppressor process that imposes a limit on the proliferative potential of normal cells that all cancer cells must bypass. Here we show by whole-genome single-nucleotide bisulfite sequencing that replicative senescent human cells exhibit widespread DNA hypomethylation and focal hypermethylation. Hypomethylation occurs preferentially at gene-poor, late-replicating, lamin-associated domains and is linked to mislocalization of the maintenance DNA methyltransferase (DNMT1) in cells approaching senescence. Low-level gains of methylation are enriched in CpG islands, including at genes whose methylation and silencing is thought to promote cancer. Gains and losses of methylation in replicative senescence are thus qualitatively similar to those in cancer, and this 'reprogrammed' methylation landscape is largely retained when cells bypass senescence. Consequently, the DNA methylome of senescent cells might promote malignancy, if these cells escape the proliferative barrier.

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Figures

Figure 1
Figure 1
Senescent cells exhibit overall hypomethylation and focal hypermethylation. (a) Proliferating (Prolif.) and senescent (Sen.) IMR90 cells stained for 5-methylcytosine (5-MeC) and single-stranded DNA(ssDNA). Scale bar, 5µm. (b) Quantitation of a in three independent experiments (100 cells each); error bars, mean ± s.e.m. (c) Percentages of methylated and unmethylated basecalls at reference CpG dinucleotides in proliferating and senescent cells. (d) Numbers of CpG dinucleotides becoming hypomethylated (hypo) and hypermethylated (hyper) in senescence. (e) Plot of chromosome 4 (chr 4) percentage methylation for proliferating and senescent cells. An overlay of these two plots is shown with difference p, proportional to the P value differences between methylation of proliferating and senescent cells. Negative and positive values are hypomethylation and hypermethylation, respectively. (f) Enlargement of boxed region from e. (g,h) Across the whole genome, regions of intermediate methylation in proliferating cells tend to be hypomethylated in senescence. For g and h CpGs were split into 100 bins on the basis of proliferating methylation. The average senescent methylation/methylation difference of each bin was then computed and plotted. (i) Number of hypomethylated and hypermethylated DMRs (differentially methylated regions) over whole genome in senescent cells. (j) Average size of hypomethylated and hypermethylated DMRs over whole genome. bp, base pairs. For c–j data are from whole-genome bisulfite sequencing of three independent replicates.
Figure 2
Figure 2
Promoter proximal methylation of transcriptionally repressed cell cycle genes. (a) Ln fold change of gene expression between proliferating and senescent cells (positive values, increased expression in senescence; negative values, decreased expression in senescence) against the difference in promoter methylation between proliferating and senescent cells (Sen. - Prolif.). Analysis only for genes expressed above the median level of expression in proliferating cells. The red numbers show the number of genes (expressed in proliferating cells) with indicated directions of expression (FDR<0.05 and fold change >1.5) and promoter methylation (FDR < 0.05) changes. (b,c). Example of a cluster (cluster 1) of downregulated genes with similar differential methylation signatures identified during execution of the gene list tool in ref. . (b) Cluster 1 location in a dendrogram of signatures arranged by shape. Green bars denote downregulated genes; red bars denote upregulated genes. Cluster 1 highlighted in blue and with arrow. (c) A close-up view of cluster 1, with gene names. Cluster 1 contains two replicates of the RFC2 gene. (d) Three plots on the left: differential methylation versus position up- and downstream of the transcription start site (TSS) (−5kb (kilobases) to +5kb) of RFC2 gene. The y axis is a differential methylation score that ranges from −1 to 1, denoting complete hypomethylation and hypermethylation, respectively. The vertical dashed centre line marks the TSS. Fold change (log2) gene expression is indicated in green. Data from all three replicates are shown. Rightmost plot: relative CpG density around RFC2 gene TSS, plotted in red. (e) Average plot of differential methylation (green) versus position up- and downstream of TSS (−5kb to +5kb) for all 136 downregulated genes in Supplementary Table 11 (three upregulated genes are excluded), smoothed over 100 bp sliding windows. The left axis is average methylation difference, plotted in green. Units are in differential methylation (ranging from −1 to 1, as for a). The right axis is CpG density, plotted in red. Units are CpG sites per base pair. (f) Venn diagram showing overlap of genes in Supplementary Tables 9 and 11. Overlap = 27.0-fold enrichment over random, P <0.0001.
Figure 3
Figure 3
DNA hypomethylation occurs before cell cycle exit and is associated with mislocalization of DNMT1. (a) Quantitation of 5-methylcytosine by immunofluorescence in cyclin-A-positive (cyclin A+) proliferating and near-senescent cells. See Supplementary Methods for definitions of Prolif, Near-sen and Sen. Low methylation denotes 900–1,700 average intensity units, intermediate methylation 1,800–2,200 units and high methylation 2,300–2,900 units. (b) Quantitation of 5-methylcytosine in proliferating (Prolif.), senescent (Sen.) and cells fixed 2 weeks and 4 weeks after onset of senescence, Sen.+2 wks and Sen.+4 wks respectively. (c) Western blot of DNMT1, DNMT3B and actin across indicated cell population doublings (PDs) and senescent cells. Mr (K), relative molecular mass (thousands). (d) RNA sequencing (RNA-seq) analysis showing aligned reads (fragments per bp per million mapped reads) on DNMT1 gene in proliferating and senescent cells. (e) Quantitative RT-PCR analysis of DNMT1 expression in proliferating and senescent cells normalized to expression of GAPDH as a housekeeping gene. mRNA, messenger RNA. (f) Quantitation of proliferating and near-senescent cyclin A-positive cells containing DNMT1 foci (from g). (g) Immunofluorescence of DNMT1 and cyclin A in proliferating (Prolif) and near-senescent (Near-sen.) cells. Arrowheads mark DNMT1 puncta. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 5 µm. (h) Immuno-FISH of satellite 2 and DNMT1 with merge: DNMT1, red; satellite 2, green. Scale bar, 5µm. (i) Immunofluorescence in situ hybridization (immuno-FISH) of satellite 2, PCNA and merge: PCNA, red; satellite 2, green. Scale bar, 5 µm. (j) Quantitation of proliferating and senescent cyclin A-positive cells that contain PCNA foci. Source data for a,b,e,f,j in Supplementary Table 22. Uncropped images of blots/gels are shown in Supplementary Fig. 8e.
Figure 4
Figure 4
Hypomethylation and expression of late-replicating satellite sequences in senescent cells. (a) Bisulfite sequencing of satellite 2 in proliferating and senescent cells. Each row represents an individual clone. Filled and open circles are methylated CpG and unmethylated CpG dinucleotides respectively. (b) Southern blot of genomic DNA with the methyl-sensitive enzyme BstBI probed with a probe specific for satellite 2. Genomic DNA was harvested at different timepoints as cells approach senescence as indicated by arrow. (c) FISH using a probe for satellite 2 in proliferating and near-senescent cells. Scale bar, 5µm. (d) Measurement of two-dimensional (2D) area occupied by the FISH probe from c. (e) RT-PCR of satellite 2 in IMR90 cells at indicated PD and senescence. (f) FISH using a probe for MYC in proliferating and near-senescent cells as a negative control for c. Scale bar, 5µm. (g) Measurement of the two-dimensional area occupied by the MYC FISH probe in f. For all graphs, error bars indicate the mean ± s.e.m. of three independent experiments where 100 cells were scored per experiment. Source data for d,g in Supplementary Table 22.
Figure 5
Figure 5
Hypomethylation occurs at gene-poor, late-replicating and lamin-associated domains whereas hypermethylation occurs at gene-rich, early-replicating regions and CpG islands. (a) Plot of a 43.5 Mb (megabase) region of chromosome 10 (chr 10) percentage methylation of proliferating (blue) and senescent (orange) cells with difference P (Fig. 1e). Normalized enrichment of lamin B1 determined by chromatin immunoprecipitation sequencing of proliferating IMR90 cells is shown. Early- and late-replicating sequences from are annotated. Genes and CpG islands are from UCSC. (b–i) Plots showing percentage overlap in total base pairs of indicated features across the whole genome, comparing observed and random predicted values. Asterisks indicate statistical significance and P <0.001.
Figure 6
Figure 6
Methylation changes in senescence resemble those in cancer. (a) Plot of a 13.5 Mb region of chromosome 16 (chr 16) with percentage methylation in proliferating (blue) and senescent (orange) cells and difference p (Fig. 1e). Genes and CpG islands are shown. Hypomethylated and hypermethylated DMRs from colon and breast cancer, hypo. cDMR and hyper. cDMR respectively, are mapped from,,. (b–e) Plots of percentage overlap in total base pairs of indicated features across whole genome, comparing observed and random predicted values; cDMRs taken from ref. . Asterisks indicate statistical significance and P-value ≪ 0.001. (f) Increased methylation of neurogenin 1 (NEUROG1) CpG island in senescence. Plot of percentage methylated basecalls in proliferating (orange) and senescent cells (blue), at the NEUROG1 gene, from whole-genome bisulfite sequencing data of three replicates of proliferating cells and three replicates of senescent cells. The orange and blue lines show the smoothed average percentage methylated basecalls at corresponding CpGs. Individual CpGs are indicated by black ticks along the x axis. The UCSC NEUROG1 gene (blue bar (hatched, exon 1)) and CpG island (green bar) are also shown. The TSS is indicated by a vertical black arrow. The gene, chromosome and bp of the CpG island are indicated at the top left.
Figure 7
Figure 7
Altered methylation is retained in cells that bypass senescence. (a) Three replicates of IMR90 cells approaching senescence (PD 80; Supplementary Fig. 1a) were infected with a control lentivirus or a lentivirus encoding SV40 T antigen. Mr (K), relative molecular mass (thousands). (b) SV40 T antigen-expressing cells exhibit extension of lifespan (bypass). (c) DNA was collected for whole-genome bisulfite sequencing from bypass cells at PD 84. Plot of chromosome 16 (chr 16) with percentage methylation for proliferating, senescent and bypass Senescent cells. Difference P is proportional to the p value difference between methylation of proliferating and senescent cells (difference P, Sen. -Prolif.), proliferating and bypass cells (difference P, Bypass - Prolif.) and senescent versus bypass cells (difference P, Bypass - Sen.). Negative and positive values are hypomethylation and hypermethylation respectively. (d) Plot of percentage methylation in 2kb windows encompassing whole genome, comparing either bypass against proliferating cells (left) or bypass against senescent cells (right).
Figure 8
Figure 8
The altered epigenome of senescent cells might promote age-associated increase in incidence of human cancers. Our analyses and previous studies and ideas lead to the following model. Senescent cells accumulate with age in human tissues. These senescent cells harbour low-level methylation of CpG islands of tumour suppressor genes that is insufficient to silence gene expression. However, even a limited/transient escape from senescence, for example owing to genetic inactivation of PTEN,, confers further rounds of cell division that permits proliferation-dependent spreading of DNA methylation. Hence, an initial ‘seed’ of methylation in a senescent cell in an aged tissue can, in conjunction with other genetic alterations and clonal selection, grow to full CpG island hypermethylation and tumour suppressor gene silencing, thereby facilitating progression to late-life cancer. Global hypomethylation in senescent cells may also promote genome instability and dysfunction,,,–
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