miR-29 Represses the Activities of DNA Methyltransferases and DNA Demethylases

Members of the microRNA-29 (miR-29) family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1) and thymine DNA glycosylase (TDG). Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.     

Epigenetic therapy for breast cancer

Both genetic and epigenetic alterations can control the progression of cancer. Genetic alterations are impossible to reverse, while epigenetic alterations are reversible. This advantage suggests that epigenetic modifications should be preferred in therapy applications. DNA methyltransferases and histone deacetylases have become the primary targets for studies in epigenetic therapy. Some DNA methylation inhibitors and histone deacetylation inhibitors are approved by the US Food and Drug Administration as anti-cancer drugs. Therefore, the uses of epigenetic targets are believed to have great potential as a lasting favorable approach in treating breast cancer.     

8a,a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.     

C5-DNA methyltransferase inhibitors: from screening to effects on zebrafish embryo development

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.     

Rapid synthesis of new DNMT inhibitors derivatives of procainamide

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.     

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human–Arabidopsis Hybrid Cell Line

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human–Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.     

Interplay between Histone and DNA Methylation Seen through Comparative Methylomes in Rare Mendelian Disorders

DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Global Changes of 5-mC/5h-mC Ratio and Methylation of Adiponectin and Leptin Gene in Placenta Depending on Mode of Delivery

It was suggested that the epigenetic alterations of the placenta are associated with obesity, as well as the delivery mode. This study aimed to assess the effect of maternal outcome and delivery procedure on global placental DNA methylation status, as well as selected 5’-Cytosine-phosphate-Guanine-3’ (CpG) sites in ADIPOQ and LEP genes. Global DNA methylation profile in the placenta was assessed using the 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) ratio evaluated with the ELISA, followed by target gene methylation patterns at selected gene regions which were determined using methylation-specific qPCR in 70 placentas from healthy, pregnant women with single pregnancy. We found no statistically significant differences in 5-mC/5-hmC ratio between intrapartum cesarean sections (CS) and vaginal deliveries (p = 0.214), as well as between elective cesarean sections and vaginal deliveries (p = 0.221). In intrapartum cesarean sections, the ADIPOQ demethylation index was significantly higher (the average: 1.75) compared to elective cesarean section (the average: 1.23, p = 0.010) and vaginal deliveries (the average: 1.23, p = 0.011). The LEP demethylation index did not significantly differ among elective CS, intrapartum CS, and vaginal delivery groups. The demethylation index of ADIPOQ correlated negatively with LEP in the placenta in the vaginal delivery group (r = −0.456, p = 0.017), but not with the global methylation. The methylation of a singular locus might be different depending on the mode of delivery and uterine contractions. Further studies should be conducted with locus-specific analysis of the whole genome to detect the methylation index of specific genes involved in metabolism.     

Genetics and Epigenetics in Asthma

Asthma is one of the most common respiratory disease that affects both children and adults worldwide, with diverse phenotypes and underlying pathogenetic mechanisms poorly understood. As technology in genome sequencing progressed, scientific efforts were made to explain and predict asthma’s complexity and heterogeneity, and genome-wide association studies (GWAS) quickly became the preferred study method. Several gene markers and loci associated with asthma susceptibility, atopic and childhood-onset asthma were identified during the last few decades. Markers near the ORMDL3/GSDMB genes were associated with childhood-onset asthma, interleukin (IL)33 and IL1RL1 SNPs were associated with atopic asthma, and the Thymic Stromal Lymphopoietin (TSLP) gene was identified as protective against the risk to TH2-asthma. The latest efforts and advances in identifying and decoding asthma susceptibility are focused on epigenetics, heritable characteristics that affect gene expression without altering DNA sequence, with DNA methylation being the most described mechanism. Other less studied epigenetic mechanisms include histone modifications and alterations of miR expression. Recent findings suggest that the DNA methylation pattern is tissue and cell-specific. Several studies attempt to describe DNA methylation of different types of cells and tissues of asthmatic patients that regulate airway remodeling, phagocytosis, and other lung functions in asthma. In this review, we attempt to briefly present the latest advancements in the field of genetics and mainly epigenetics concerning asthma susceptibility.     

Epigenetic Regulation of Breast Cancer Stem Cells Contributing to Carcinogenesis and Therapeutic Implications

Globally, breast cancer has remained the most commonly diagnosed cancer and the leading cause of cancer death among women. Breast cancer is a highly heterogeneous and phenotypically diverse group of diseases, which require different selection of treatments. Breast cancer stem cells (BCSCs), a small subset of cancer cells with stem cell-like properties, play essential roles in breast cancer progression, recurrence, metastasis, chemoresistance and treatments. Epigenetics is defined as inheritable changes in gene expression without alteration in DNA sequence. Epigenetic regulation includes DNA methylation and demethylation, as well as histone modifications. Aberrant epigenetic regulation results in carcinogenesis. In this review, the mechanism of epigenetic regulation involved in carcinogenesis, therapeutic resistance and metastasis of BCSCs will be discussed, and finally, the therapies targeting these biomarkers will be presented.     

Influence of Benzo(a)pyrene on Different Epigenetic Processes

Epigenetic changes constitute one of the processes that is involved in the mechanisms of carcinogenicity. They include dysregulation of DNA methylation processes, disruption of post-translational patterns of histone modifications, and changes in the composition and/or organization of chromatin. Benzo(a)pyrene (BaP) influences DNA methylation and, depending on its concentrations, as well as the type of cell, tissue and organism it causes hypomethylation or hypermethylation. Moreover, the exposure to polyaromatic hydrocarbons (PAHs), including BaP in tobacco smoke results in an altered methylation status of the offsprings. Researches have indicated a potential relationship between toxicity of BaP and deregulation of the biotin homeostasis pathway that plays an important role in the process of carcinogenesis. Animal studies have shown that parental-induced BaP toxicity can be passed on to the F1 generation as studied on marine medaka (Oryzias melastigma), and the underlying mechanism is likely related to a disturbance in the circadian rhythm. In addition, ancestral exposure of fish to BaP may cause intergenerational osteotoxicity in non-exposed F3 offsprings. Epidemiological studies of lung cancer have indicated that exposure to BaP is associated with changes in methylation levels at 15 CpG; therefore, changes in DNA methylation may be considered as potential mediators of BaP-induced lung cancer. The mechanism of epigenetic changes induced by BaP are mainly due to the formation of CpG-BPDE adducts, between metabolite of BaP—BPDE and CpG, which leads to changes in the level of 5-methylcytosine. BaP also acts through inhibition of DNA methyltransferases activity, as well as by increasing histone deacetylases HDACs, i.e., HDAC2 and HDAC3 activity. The aim of this review is to discuss the mechanism of the epigenetic action of BaP on the basis of the latest publications.     

DNA甲基化对调控胰岛分化基因表达的影响

目的探讨DNA甲基化对调控胰岛分化基因表达的影响,为调控干细胞分化为胰岛细胞提供理论依据。方法采用甲基化DNA免疫共沉淀-实时定量PCR法(MeDIP-qPCR)检测129/J小鼠胚胎干细胞、NIT1细胞及NIH3T3细胞中Pdx-1、MafA、Nkx6.1和Oct4四种调控胰岛分化基因的DNA甲基化程度;同时采用实时定量RT-PCR检测上述3种细胞中4种基因mRNA表达水平,分析这些基因DNA甲基化水平差异与基因表达之间的关系。结果 Pdx-1、MafA和Nkx6.1基因在129/J小鼠胚胎干细胞和NIT1细胞中呈低甲基化,在NIH3T3细胞中则呈高甲基化,前两种细胞的甲基化程度明显低于后者(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中未甲基化,在NIT1和NIH3T3细胞中呈低甲基化,NIT1细胞的甲基化程度明显低于NIH3T3细胞(P<0.05)。低甲基化的Pdx-1、MafA和Nkx6.1基因在NIT1细胞中可高效表达,而在NIH3T3和129/J小鼠胚胎干细胞中则未见表达(P<0.05);Oct4基因在129/J小鼠胚胎干细胞中可高效表达,在NIT1和NIH3T3细胞中则未见表达。结论转录起始区DNA甲基化程度可影响Pdx-1、MafA、Nkx6.1和Oct4基因的表达,参与β细胞的分化过程。     

Epigenetic effects of environmental chemicals bisphenol a and phthalates

The epigenetic effects on DNA methylation, histone modification, and expression of non-coding RNAs (including microRNAs) of environmental chemicals such as bisphenol A (BPA) and phthalates have expanded our understanding of the etiology of human complex diseases such as cancers and diabetes. Multiple lines of evidence from in vitro and in vivo models have established that epigenetic modifications caused by in utero exposure to environmental toxicants can induce alterations in gene expression that may persist throughout life. Epigenetics is an important mechanism in the ability of environmental chemicals to influence health and disease, and BPA and phthalates are epigenetically toxic. The epigenetic effect of BPA was clearly demonstrated in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and the hypomethylating effect of BPA was prevented by maternal dietary supplementation with a methyl donor like folic acid or the phytoestrogen genistein. Histone H3 was found to be trimethylated at lysine 27 by BPA effect on EZH2 in a human breast cancer cell line and mice. BPA exposure of human placental cell lines has been shown to alter microRNA expression levels, and specifically, miR-146a was strongly induced by BPA treatment. In human breast cancer MCF7 cells, treatment with the phthalate BBP led to demethylation of estrogen receptor (ESR1) promoter-associated CpG islands, indicating that altered ESR1 mRNA expression by BBP is due to aberrant DNA methylation. Maternal exposure to phthalate DEHP was also shown to increase DNA methylation and expression levels of DNA methyltransferases in mouse testis. Further, some epigenetic effects of BPA and phthalates in female rats were found to be transgenerational. Finally, the available new technologies for global analysis of epigenetic alterations will provide insight into the extent and patterns of alterations between human normal and diseased tissues. In vitro models such as human embryonic stem cells may be extremely useful in bettering the understanding of epigenetic effects on human development, health and disease, because the formation of embryoid bodies in vitro is very similar to the early stage of embryogenesis.