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Summary 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.

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 tRNAAsp, 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. [Cancer Res 2009;69(20):8127–32]

In this paper, we compare the advertisement calls of 207 neotropical strawberry poison frogs (Dendrobates pumilio) collected in 21 localities along a transect from northern Costa Rica to western Panama. Populations varied most in call duration and call rate, while pulse rate and duty cycle were less variable. Multivariate analyses showed that call variation followed a cline with higher call rates, shorter calls, lower duty cycles and higher pulse rates in the southeast. Body size decreased towards the southeast and explained most variation in dominant frequency, as well as some residual variation in call rate. We conclude that a combination of geography and morphology is largely responsible for call variation within this species. Two inferred bio-acoustic groups were roughly in accordance with two genetic groups, geographically separated in central Costa Rica. However, genetic distances among populations did not co-vary with call dissimilarity after correction for geographic distances. Thus, differences in calls between genetic groups are probably mainly a result of clinal variation. These findings agree with the general observation that bio-acoustic variation is often not (highly) associated with genetic divergence. Moreover, colour polymorphism observed among Panamanian populations was not reflected in a higher variability in call parameters relative to the monomorphic Costa Rican populations.

to the blind-ending lymphatic vasculature, the blood vasculature represents a closed circulating system and facilitates the exchange of gases (i.e., O 2 and CO 2 ) and nutrients [1]. Although both BECs and LECs display distinct features and functions, they share a close molecular and developmental relationship. In the early embryo, progenitor cells, the so-called angioblasts, differentiate from the mesodermal compartment and establish a primitive vascular network [2,3]. After determination of arteries and veins, venous endothelial cells further transdifferentiate into LECs forming primary lymph sacs [1]. This process is carefully regulated and requires a defined expression of lineage-specifying genes (i.e., PROX1) [4]. After terminal endothelial differentiation both LECs and BECs are characterized by distinct gene-expression profiles regulating cell type-specific functions [5]. There is growing evidence to suggest that epigenetic mechanisms (i.e., DNA methylation at CpG dinucleotides and histone modifications) are involved in regulating vascular endothelial gene-expression changes during development and in maintaining cell-type-specific expression patterns in adulthood. For example, histone deacetylase activity has been implicated in the regulation of embryonic endothelial differentiation [6], and histone acetylation controls the expression of NOTCH4 [7], a key signaling molecule involved in vascular development and remodeling. Furthermore, shear stress, which is a particularly distinctive stimulus for vessels and vessel-associated diseases, can alter chromatin structure and change the gene-expression profile in cultured human endothelial cells [8]. In addition, histone modifications such as H3K4 methylation are involved in vessel sprouting and endothelial cell migration [9]. However, the role of Nearly all cells of the human body have an identical genetic background. Nevertheless, cellular specialization into distinct functional entities is a hallmark of biological complexity in multi cellular organisms. But how can a single stem cell give rise to a variety of functionally different cell types? During past decades it has become clear that epigenetic mechanisms play a crucial role in regulating cell type-specific gene expression and thereby contribute to the determination of cellular fate. Furthermore, alterations of epigenetic signatures cause a variety of diseases. The fact that epigenetic marks are reversible makes them an attractive target for therapeutic interventions, aiming at reconstituting a healthy phenotype by reversing aberrant methylation marks. The present editorial summarizes the latest findings on epigenetic regulation and disease-associated dysregulation of blood and lymphatic vessel formation, focusing primarily on the characterization of blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Although both cell types share a close developmental relationship, they show specific functional differences. Thus, BECs and LECs represent an interesting model system to investigate the involvement of epigenetic mechanisms in regulating these cell-type-specific functions. We will further discuss whether treatment approaches targeting aberrant methylation marks may represent a rewarding strategy to recover epigenetically induced functional disorders of the vascular systems. The human vasculature is characterized by two types of tubular networks; the blood and the lymphatic systems, which comprise different but interdependent functions. The lymphatic system regulates tissue fluid homeostasis, immune surveillance, and absorption of fluids and macromolecules from the interstitium [1]. In contrast

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.

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.

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.

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.

Silencing of genes by DNA 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 after depletion of DNMT3B protein have not been analyzed in detail yet. We performed short- and long-term RNAi knockdown experiments to reduce DNMT3B protein levels in colon cancer cell lines and analyzed genome-wide DNA methylation changes on HumanMethylation27 and HumanMethylation450 Illumina bead chips. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. All colon cancer cell lines tested contain a mutant K-Ras and we conclude that K-Ras status - in contrast to DNMT3B protein overexpression levels - does not impact on anti-proliferative responses after DNMT3B knockdown. However, in contrast to the dramatic alterations of DNA methylation observed in DNMT1; DNMT3B double knockout cells (DKO), 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1036. doi:1538-7445.AM2012-1036

We describe primers and PCR conditions to amplify nine new tetranucleotide loci and one new dinucleotide locus isolated from the strawberry poison frog (Oophaga pumilio). In 21 individuals from Costa Rica, the number of alleles ranged from 4 to 16, observed heterozygosities from 40 to 100%, and polymorphic information content ranged from 0.60 to 0.90 per locus. Evidence for linkage disequilibrium was found only between two loci, but this pattern was not found in other populations tested. All primer pairs cross-amplified in Oophaga vicentei from Panama.

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.