H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors

Pearson correlation of biological characteristics with donor age.

*<p>represents p<0.05.</p

Correlation between biological parameters and donor age.

<p>A correlation between age and clinical and biological labels could not be determined for labels such as A) population doublings per day between day 0–1 and bone formation. Correlation with donor age could solely be determined for B) the mean ALP expression and C) the percentage ALP positive cells in basic medium.</p

Gene-expression correlated to <i>in vitro</i> aging.

<p>The trend observed in relation to <i>in vivo</i> aging could not be confirmed in hMSCs aged <i>in vitro</i>. Gene-expression was determined up to passage 7 in three donors (D.024: female, 59 years, acetabulum; D.036: female, 38 years, iliac crest; D.118A: male, 60 years, acetabulum), however a general trend could not be observed. For some genes the expression pattern was random, A) <i>COL13A1</i> and D) <i>ZNF395</i>, and for other genes there seemed to be a trend in one of the donors, but this could not be confirmed in the other two, B) <i>FST</i> and C) <i>JAG1</i>.</p

qPCR primer sequences.

<p>qPCR primer sequences.</p

List of genes correlating in hMSCs with donor age (p<0.01) based on ANOVA.

a<p>: p values for genes correlating with age corrected for the confounding effects attributed to the sex of the donor and the aspiration site of the bone marrow. The p values were computed omitting the samples with unknown origin.</p>b<p>: FDR = the false discovery rate.</p>c<p>: The slope corresponds to the strength of the correlation. This value can be considered as the fold change per year. (i.e. 0.017 corresponds to a fold change of 1,7 per 100 years).</p>d<p>: The genes presented in bold are affected by the aspiration site of the bone marrow (acetabulum or iliac crest) (p<0.01).</p

Gene expression validated with qPCR in 20 donors.

*<p>represents p<0.05.</p>**<p>R is the Pearson correlation value with donor age.</p>#<p>validated on 61 donors (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042908#pone-0042908-g003" target="_blank">Figure 3</a>).</p

GO Enrichment analysis with enrichment for all three GO categories.

<p>The GO term and name are given, along with the total number of genes correlating with age in the category and fold enrichment (FE) of the GO term. For each GO category, the enriched categories or the top 10 is presented.</p>a<p>Fold Enrichment of the GO term in the top list of genes (p<0.01; as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042908#pone-0042908-t002" target="_blank">Table 2</a>).</p>b<p>Number of genes.</p

qPCR validation on the whole donor population.

<p>Correlation curves were produced for the expression of A) <i>COLEC12</i>, B) <i>COL13A1</i>, C) <i>FGFR2</i>, D) <i>FST</i>, E) <i>JAG1</i>, F) <i>SLIT3</i> and G) <i>ZNF395</i> in the entire donor population and significant correlations could be confirmed.</p

Validation of gene-expression in rat MSCs.

<p>To assess if age-related gene-expression was similar inter-species, MSCs were isolated from 1, 12 and 24 month old rats. The correlation between the expression of follistatin and age could be verified in rat MSCs, suggesting this is possibly a marker for several species. For the other three genes no correlation was found.</p