Reduced DNA methylation patterning and transcriptional connectivity define human skin aging.

Epigenetic changes represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Age-related changes in DNA methylation at the genome scale have been termed 'epigenetic drift', but the defining features of this phenomenon remain to be established. Human epidermis represents an excellent model for understanding age-related epigenetic changes because of its substantial cell-type homogeneity and its well-known age-related phenotype. We have now generated and analyzed the currently largest set of human epidermis methylomes (N = 108) using array-based profiling of 450 000 methylation marks in various age groups. Data analysis confirmed that age-related methylation differences are locally restricted and characterized by relatively small effect sizes. Nevertheless, methylation data could be used to predict the chronological age of sample donors with high accuracy. We also identified discontinuous methylation changes as a novel feature of the aging methylome. Finally, our analysis uncovered an age-related erosion of DNA methylation patterns that is characterized by a reduced dynamic range and increased heterogeneity of global methylation patterns. These changes in methylation variability were accompanied by a reduced connectivity of transcriptional networks. Our findings thus define the loss of epigenetic regulatory fidelity as a key feature of the aging epigenome.

Epigenetic regulation of depot-specific gene expression in adipose tissue.

In humans, adipose tissue is distributed in subcutaneous abdominal and subcutaneous gluteal depots that comprise a variety of functional differences. Whereas energy storage in gluteal adipose tissue has been shown to mediate a protective effect, an increase of abdominal adipose tissue is associated with metabolic disorders. However, the molecular basis of depot-specific characteristics is not completely understood yet. Using array-based analyses of transcription profiles, we identified a specific set of genes that was differentially expressed between subcutaneous abdominal and gluteal adipose tissue. To investigate the role of epigenetic regulation in depot-specific gene expression, we additionally analyzed genome-wide DNA methylation patterns in abdominal and gluteal depots. By combining both data sets, we identified a highly significant set of depot-specifically expressed genes that appear to be epigenetically regulated. Interestingly, the majority of these genes form part of the homeobox gene family. Moreover, genes involved in fatty acid metabolism were also differentially expressed. Therefore we suppose that changes in gene expression profiles might account for depot-specific differences in lipid composition. Indeed, triglycerides and fatty acids of abdominal adipose tissue were more saturated compared to triglycerides and fatty acids in gluteal adipose tissue. Taken together, our results uncover clear differences between abdominal and gluteal adipose tissue on the gene expression and DNA methylation level as well as in fatty acid composition. Therefore, a detailed molecular characterization of adipose tissue depots will be essential to develop new treatment strategies for metabolic syndrome associated complications.

Aging is associated with highly defined epigenetic changes in the human epidermis.

BACKGROUND: Altered DNA methylation patterns represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Several studies have described global and complex age-related methylation changes, but their structural and functional significance has remained largely unclear. RESULTS: We have used transcriptome sequencing to characterize age-related gene expression changes in the human epidermis. The results revealed a significant set of 75 differentially expressed genes with a strong functional relationship to skin homeostasis. We then used whole-genome bisulfite sequencing to identify age-related methylation changes at single-base resolution. Data analysis revealed no global aberrations, but rather highly localized methylation changes, particularly in promoter and enhancer regions that were associated with altered transcriptional activity. CONCLUSIONS: Our results suggest that the core developmental program of human skin is stably maintained through the aging process and that aging is associated with a limited destabilization of the epigenome at gene regulatory elements.

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation.

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.

DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells.

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines.

The cytosine analogues azacytidine and decitabine are currently being developed as drugs for epigenetic cancer therapy. Although various studies have shown that both drugs are effective in inhibiting DNA methylation, it has also become clear that their mode of action is not limited to DNA demethylation. Because azacytidine is a ribonucleoside, the primary target of this drug may be cellular RNA rather than DNA. We have now analyzed the possibility that azacytidine inhibits the RNA methyltransferase DNMT2. We found that DNMT2 is variably expressed in human cancer cell lines. RNA bisulfite sequencing showed that azacytidine, but not decitabine, inhibits cytosine 38 methylation of tRNA(Asp), a major substrate of DNMT2. Azacytidine caused a substantially stronger effect than decitabine on the metabolic rate of all the cancer cell lines tested, consistent with an effect of this drug on RNA metabolism. Of note, drug-induced loss of RNA methylation seemed specific for DNMT2 target sites because we did not observe any significant demethylation at sites known to be methylated by other RNA methyltransferases. Our results uncover a novel and quantifiable drug activity of azacytidine and raise the possibility that tRNA hypomethylation might contribute to patient responses.