A genomic predictor of lifespan in vertebrates

Gene promoters are evolutionarily conserved across holozoans and enriched in CpG sites, the target for DNA methylation. As animals age, the epigenetic pattern of DNA methylation degrades, with highly methylated CpG sites gradually becoming demethylated while CpG islands increase in methylation. Across vertebrates, aging is a trait that varies among species. We used this variation to determine whether promoter CpG density correlates with species’ maximum lifespan. Human promoter sequences were used to identify conserved regions in 131 mammals and a subset of 28 primate genomes. We identified approximately 1000 gene promoters (5% of the total), that significantly correlated CpG density with lifespan. The correlations were performed via the phylogenetic least squares method to account for trait similarity by common descent using phylogenetic branch lengths. Gene set enrichment analysis revealed no significantly enriched pathways or processes, consistent with the hypothesis that aging is not under positive selection. However, within both mammals and primates, 95% of the promoters showed a positive correlation between increasing CpG density and species lifespan, and two thirds were shared between the primate subset and mammalian datasets. Thus, these genes may require greater buffering capacity against age-related dysregulation of DNA methylation in longer-lived species.

Decision letter: Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

Simultaneous treatment of cancer cells with DNA methyltransferase and HDAC inhibitors leads to synergistic increases of gene expression, indicating that certain genes require targeting of multiple modes of epigenetic regulation to be fully reactivated. However, a major obstacle facing the use of epigenetic therapies such as HDAC inhibitors in cancer is the lack of specificity in reactivating gene expression. Combined targeting of DNA and histone methylation is an unexplored and promising alternative to current therapies due to the role of both modifications in silencing of various tumor suppressor genes in cancer. To address this possibility, we performed a genome wide gene expression study of a DNA methyltransferase inhibitor (DAC) in combination with a LSD1 inhibitor (S2101), G9a inhibitor (UNC0638), EZH2 inhibitor (GSK343) or a HDAC inhibitor (Depsipeptide) in colon cancer (SW48) and breast cancer (MCF7) models. The trends of gene expression changes in response to the inhibitors were similar in both cancer models. S2101, UNC0638, and GSK343 treatment by themselves induced limited gene upregulation in comparison to DAC and especially Depsipeptide. Interestingly, UNC0638 along with DAC had a strong preference to upregulate genes that had high levels of DNA methylation in their promoters. DAC in combination with S2101, UNC0638, or GSK343 was able to synergistically or additively upregulate many DAC target genes compared to levels achieved by DAC alone, demonstrated limited overlap between the upregulated genes (only 4.42% of genes commonly upregulated in SW48, 0.32% in MCF7), and formed distinct clusters in a hierarchical cluster analysis. Each DAC+inhibitor combination also induced a significant number of novel genes that were only upregulated with the combination therapy and not with DAC or inhibitor alone (>490 genes for each inhibitor combination in both cancer models), and there was again limited overlap between the upregulated genes (only 7.64% of genes commonly upregulated in SW48, 7.31% in MCF7). IPA analysis demonstrated that genes induced uniquely by the inhibitor combinations are enriched in critical cancer regulation pathways such as cell proliferation, cell death, and cell movement. Overall, these results demonstrate that effectiveness of combined targeting of DNA and histone methylation is achieved through two mechanisms: 1) Increase effectiveness of DAC by further upregulation of DAC target genes, and 2) Reactivate novel target genes unable to be induced by DAC or the inhibitors alone. These results also demonstrate a promising ability to target specific, non-overlapping sets of genes through different inhibitor combinations, contrary to the nonspecific effect observed with traditional epigenetic therapies. Combined targeting of DNA and histone methylation is a promising novel epigenetic therapy that may allow for the targeting of specific tumor types based on their gene expression profiles. Citation Format: Takahiro Sato, Matteo Cesaroni, Jaroslav Jelinek, Jean-Pierre Issa. Combined targeting of DNA and histone methylation improves the efficacy and specificity of epigenetic therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3521. doi:10.1158/1538-7445.AM2015-3521

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects invitro and invivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

BackgroundTumour suppressor genes are often transcriptionally silenced by promoter hypermethylation, and recent research has implicated alterations in chromatin structure as the mechanistic basis for this repression. In addition to DNA methylation, other epigenetic post-translational modifications that modulate the stability and binding of specific transcription factors to gene promoters have emerged as important mechanisms for controlling gene expression. The aim of this study was to analyse the implications of these mechanisms and their molecular connections in the reactivation of RASSF1A in breast cancer.MethodsCompounds that modulate the intracellular concentration of adenosine, such as dipyridamole (DIPY), greatly increase the antiproliferative effects of 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin (TMCG), a synthetic antifolate derived from the structure of tea catechins. Quantitative real-time PCR arrays and MALDI-TOF mass spectrometry indicated that this combination (TMCG/DIPY) induced apoptosis in breast cancer cells by modulating the methylation levels of DNA and proteins (such as E2F1), respectively. Chromatin immunoprecipitation (ChIP) assays were employed to confirm that this combination induced chromatin remodelling of the RASSF1A promoter and increased the occupancy of E2F1 at the promoter of this tumour suppressor gene.ResultsThe TMCG/DIPY combination acted as an epigenetic treatment that reactivated RASSF1A expression and induced apoptosis in breast cancer cells. In addition to modulating DNA methylation and chromatin remodelling, this combination also induced demethylation of the E2F1 transcription factor. The ChIP assay showed enhancement of E2F1 occupancy at the unmethylated RASSF1A promoter after TMCG/DIPY treatment. Interestingly, inhibition of E2F1 demethylation using an irreversible inhibitor of lysine-specific demethylase 1 reduced both TMCG/DIPY-mediated RASSF1A expression and apoptosis in MDA-MB-231 cells, suggesting that DNA and protein demethylation may act together to control these molecular and cellular processes.Conclusions/SignificanceThis study demonstrates that simultaneous targeting of DNA and E2F1 methylation is an effective epigenetic treatment that reactivates RASSF1A expression and induces apoptosis in breast cancer cells.

Dna-Methylation Signature of Microrna Coding Genes Associated with Chronological Age in a High-Cardiovascular Risk Mediterranean Population. Sex-Specific Analysis

DNA methylation is an ancient epigenetic pathway found across eukaryotes. Nevertheless, the targets of DNA methylation within genomes evolve extremely rapidly. Arthropods display many such examples. The mealybug Planococcus citri has evolved methylation at promoter sequences, associated with gene silencing just as in mammals. In this issue of Molecular Ecology, Bain et al. (2021), thoroughly characterise mealybug methylation, exploring its potential functions in gene expression and the spectacular sexual dimorphism that is a characteristic of this species. Their results provide new insights into the complex relationship between DNA methylation and gene expression and highlight how rapidly different methylation systems can evolve.

Histone de-acetylases and epigenetic regulation

The influence of increasing concentrations of heavy metals solutions (Pb(NO3)2, CuSO4, ZnSO4) on the modification of content of proline in the plants Cannabis sativa L was studied. It is supposed that accumulation of amino acid in the plants has an adaptive character concerning stress factors. The dependence of modification of the level of free proline from concentrations of salt in the medium of cultivation was exposed.

Endometriosis (EMS) is a gynecological disease defined by the translocation and growth of endometrial tissue in other tissues or organs outside the uterus. Its clinical manifestations are dysmenorrhea, irregular menstruation, and even infertility. Although EMS is a benign disease, it has the characteristics of malignant tumor and the potential of malignant transformation. Recent studies have found that EMS may involve epigenetic changes and that various epigenetic aberrations, especially aberrant DNA methylation may play an essential role in the pathogenesis of EMS. Previous studies have elucidated the epigenetic regulators of EMS and reported variations in epigenetic patterns of genes known to be associated with abnormal hormonal, immune, and inflammatory states of EMS. With the development of high-throughput sequencing and other biomolecular technologies, we have a better understanding of genome-wide methylation in EMS. This article will discuss the potentiality of targeting DNA methylation as the therapeutic approach for EMS. This article reviews the role of DNA methylation in the pathophysiology of EMS and provides insight into a novel therapeutic approach for EMS by targeting DNA methylation modifiers. We also review the current progress in using DNA methylation inhibitors in EMS therapy and the potential promise and challenges ahead. Aberrant DNA methylation plays an essential role in the pathogenesis of EMS and epigenetic agents targeting DNA methyltransferases are expected to be the theoretical basis for the new treatment of EMS.

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

The Neuroepigenetics of Suicide

Epigenetic modifications play a crucial role in gene regulation and have been implicated in various physiological processes and disease conditions. DNA methylation (DNAm) has been implicated in the etiology and progression of many stress-related psychiatric behaviors, such as depression. The ability to manipulate DNAm may provide a means to reverse and treat such disorders. Although CRISPR-based technologies have enabled locus-specific DNAm editing, their clinical applicability may be limited due to immunogenicity concerns and off-target effects. In this study, we introduce a novel approach for targeted DNAm manipulation using single-stranded methylated DNA probes. The probes were designed against the GRE of FKBP5 and the promoter region of MAOA. In both human embryonic kidney HEK293 and mouse pituitary AtT-20 cells, transfection with their respective methylated probes significantly increased DNAm at targeted CpG sites in a persistent and dose-dependent manner. Importantly, the induced methylation effectively attenuated glucocorticoid-induced upregulation of FKBP5 gene expression. Alteration of methylation was specific to single-stranded probes, as double-stranded methylated probes and unmethylated probes showed no significant effects. Some limitations include the need to further characterize factors that influence probe efficiency, such as probe length and CpG density; develop an efficient in vivo probe delivery system; and perform a more extensive consideration of possible off-target effects. Despite these limitations, our findings suggest that methylated DNA probes have the potential to function as a simple tool for targeted epigenetic manipulation and serve as a safer alternative to CRISPR-based epigenome editing tools for the treatment of stress-related disorders such as depression.

Dysregulated microRNA (miRNA) expression contributes to the pathogenesis of hematopoietic malignancies, including chronic lymphocytic leukemia (CLL). However, an understanding of the mechanisms that cause aberrant miRNA transcriptional control is lacking. In this study, we comprehensively investigated the role and extent of miRNA epigenetic regulation in CLL. Genome-wide profiling conducted on 24 CLL and 10 healthy B cell samples revealed global DNA methylation patterns upstream of miRNA sequences that distinguished malignant from healthy cells and identified putative miRNA promoters. Integration of DNA methylation and miRNA promoter data led to the identification of 128 recurrent miRNA targets for aberrant promoter DNA methylation. DNA hypomethylation accounted for more than 60% of all aberrant promoter-associated DNA methylation in CLL, and promoter DNA hypomethylation was restricted to well-defined regions. Individual hyper- and hypomethylated promoters allowed discrimination of CLL samples from healthy controls. Promoter DNA methylation patterns were confirmed in an independent patient cohort, with 11 miRNAs consistently showing an inverse correlation between DNA methylation status and expression level. Together, our findings characterize the role of epigenetic changes in the regulation of miRNA transcription and create a repository of disease-specific promoter regions that may provide additional insights into the pathogenesis of CLL.