DNA Methyltransferase Family Research Articles

Heterozygous Dnmt3a Mutation Induces Expansion of Hematopoietic Stem Cell Pool in a Murine Model

Somatic mutations in DNMT3A, a member of the DNA methyltransferase family, have been identified in various kinds of hematologic malignancies including acute myeloid leukemia (AML), acute lymphoblastic leukemia, myeloproliferative neoplasms, and aplastic anemia. Especially, in cytogenetically normal AML, DNMT3A mutations are detected in 15-20% of cases and associated with poor prognosis. Approximately 40 to 60% of DNMT3A mutations reside at Arg882 (R882), forming hot spot mutation site, implying gain-of-functional property of the mutation in development of leukemia. So far, a functional role of DNMT3A mutation in leukemogenesis has been investigated by overexpressing DNMT3A R882 mutant or Dnmt3a knockout in murine hematopoietic cells. However, the consequenses of DNMT3A R882 mutant with endogenous expression have been largely unknown. To elucidate this, we generated a novel mouse model for Cre-mediated conditional expression of Dnmt3a R878C mutant (homologous to human R882C) from the endogenous locus of Dnmt3a. For hematopoietic cell-specific mutation in vivo, we crossed these mice with Vav-Cre mice. By analyzing genotypes of offspring derived from the pair of Dnmt3aR878C/wt; Vav-Crewt/wt mice and Dnmt3aR882C/wt; Vav-Cretg/wt mice, it is revealed that mice harboring homozygous mutation of Dnmt3a in hematopoietic cells (DNMT3AR882C/R882C; Vav-Cretg/wt) were not born for now. In contrast, Dnmt3aR882C/wt; Vav-Cretg/wt (hereafter Dnmt3a R878C) mice were normally born and they did not show any hematological or other disorders at least until 40 weeks after birth. Additionally, frequencies of peripheral blood mature cells in each lineage are not altered in Dnmt3a R878C mice compared to Dnmt3awt/wt Vav-Cretg/wt (hereafter control mice). To further investigate the effect of DNMT3A R882 mutation, we sacrificed Dnmt3a R878C and control mice at 8-12 weeks after birth and compared the distribution of hematopoietic cells by using flow cytometry. Although there was no obvious difference in the number of mononuclear cells in the whole bone marrow, we found a significant increase of the frequency of long-term hematopoietic stem cells (defined by CD150+ CD48- Lineage- c-Kit+ Sca-1+) in Dnmt3a R878C mice compared to control mice (0.040% and 0.019%, p = 0.022). The frequencies of other progenitors including short-term hematopoietic stem cells, multipotent progenitors, or Lineage- c-Kit+ Sca-1+ (LSK) cells were not changed between Dnmt3a R878C mice and control mice. Moreover, in order to determine whether DNMT3A mutation leads to a qualitative difference in HSCs, we performed colony forming assay. While control LSK cells could not form colonies at fifth round of replating, Dnmt3a R878C LSK cells could be serially replated up to five passages. Additionally, the numbers of colonies in each round were much higher in Dnmt3a R878C cells compared to control cells, suggesting HSCs with heterozygous Dnmt3a R878C mutation have aberrantly enhanced self-renewal capacity. Currently, we are performing competitive bone marrow transplantation assay to evaluate the functional role of DNMT3A mutation in vivo. We transplanted 500,000 bone marrow cells from Dnmt3a R878C or control mice, which are Ly5.2 background, together with the same number of Ly5.1-horboring competitor cells into Ly5.1-harboring lethally-irradiated recipient mice. Thus far, Peripheral blood chimerisms of donor cells have tended to be higher in recipients of Dnmt3a R878C mutant cells compared to control mice, suggesting that heterozygous Dnmt3a R878C mutation contribute to expansion of hematopoietic stem cell pool. Collectively, heterozygous Dnmt3a R878C mutant enhanced self-renewal capacity of HSCs and resulted in accumulation of HSCs in vivo, similar to mice models with overexpression of DNMT3A R882 mutants. Our results revealed that endogenous expression of DNMT3A mutant is sufficient for expansion of HSCs, revealing functional contribution of DNMT3A mutation in forming the backbone of premalignant status of HSCs. DisclosuresNo relevant conflicts of interest to declare.

Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation

BackgroundThe oncogenic transcription factor MYC is pathologically activated in many human malignancies. A paradigm for MYC dysregulation is offered by Burkitt lymphoma, where chromosomal translocations leading to Immunoglobulin gene-MYC fusion are the crucial initiating oncogenic events. However, Burkitt lymphoma cases with no detectable MYC rearrangement but maintaining MYC expression have been identified and alternative mechanisms can be involved in MYC dysregulation in these cases.MethodsWe studied the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level. Data was validated at the mRNA and protein level by quantitative Real-Time polymerase chain reaction and immunohistochemistry, respectively.ResultsWe identified four microRNAs differentially expressed between the two groups. The impact of these microRNAs on the expression of selected genes was then investigated. Interestingly, in MYC translocation-negative cases we found over-expression of DNA-methyl transferase family members, consistent to hypo-expression of the hsa-miR-29 family. This finding suggests an alternative way for the activation of lymphomagenesis in these cases, based on global changes in methylation landscape, aberrant DNA hypermethylation, lack of epigenetic control on transcription of targeted genes, and increase of genomic instability. In addition, we observed an over-expression of another MYC family gene member, MYCN that may therefore represent a cooperating mechanism of MYC in driving the malignant transformation in those cases lacking an identifiable MYC translocation but expressing the gene at the mRNA and protein levels.ConclusionsCollectively, our results showed that MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases are slightly different in terms of microRNA and gene expression. MYC translocation-negative Burkitt lymphoma, similarly to other aggressive B-cell non Hodgkin’s lymphomas, may represent a model to understand the intricate molecular pathway responsible for MYC dysregulation in cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1661-7) contains supplementary material, which is available to authorized users.

Effect of DNMT inhibitor on bovine parthenogenetic embryo development

DNA methylation catalyzed by DNA methyltransferase (DNMT) family plays an important role during mammal preimplanted embryo development. However, the effects of RG108, a DNMT inhibitor (DNMTi), on DNMT in the development of bovine preimplanted embryos are not fully elucidated. In this study, we investigated the role of RG108 on the development, dynamics of gene-specific DNA methylation and transcription of bovine parthenogenetic preimplantation embryos. We found that Dnmt1 and Dnmt3b showed highly transcription in parthenogenetic 2-cell embryos, and then the transcription levels decreased during the following development stages, whereas Dnmt3a was always maintained at a lower transcription level during bovine parthenogenetic preimplantation embryo development. Treatment with RG108 blocked the development of bovine parthenogenetic preimplantation embryos and induced hypomethylation in the embryos. RG108 decreased the methylation level of the Nanog gene promoter region, which caused activation of the Nanog gene in 8-cell embryos and increased the transcription level. RG108 also induced the hypomethylation of the repeat elements (satellite I and α-satellite), which may cause genome instability, increasing the number of apoptotic cells in the blastocysts and also the transcription level of the apoptotic gene Bax. These results indicate that RG108, a DNMT inhibitor (DNMTi), inhibits the development of bovine parthenogenetic preimplantation embryos, suggesting that the DNMT is necessary for bovine parthenogenetic preimplanatation embryo development.

Unique Role for a DNA Methyltransferase Isoform in Lung Cancer

Aberrant DNA methylation patterns are one of the most studied epigenetic components in cancer development. In certain cancers, altered expression of DNA methyltransferase (DNMT) family members could potentially cause this DNA methylation re-patterning. Specifically, DNMT3B has over thirty different isoforms with varied expression in tissues (Ostler et al. 2007). Recent studies have shown that DNMT3B is responsible for gene body methylation by recognizing the H3K36me3 modification found in gene bodies, especially gene body remethylation after treatment with a DNA methylation inhibitor (5-Aza-CdR) (Baubec et al., 2015, Yang et al., 2014). The methylated gene bodies showed positive correlations with gene expression and, additionally, hypomethylation of gene bodies could lead to down regulation of gene expression (Yang et al. 2014). Non-small cell lung cancer (NSCLC) is one such disease which has overexpression of DNMT3B, including a specific subfamily which lacks an N-terminal domain (ΔDNMT3B) (Wang et al. 2006). In this issue of EBioMedicine the work presented by Ma et al. (Ma et al. 2015), provides exciting developments for NSCLC with findings connecting the aberrant DNA methylation patterns during tumorigenesis with the predominantly expressed DNMT3B isoform: ΔDNMT3B4-del, a truncated DNMT3B isoform lacking exons 21 and/or 22 which contain the catalytic domain.

DNA methyltransferases have an essential role in female fecundity in brown planthopper, Nilaparvata lugens

DNA methylation is an ancient epigenetic modification present in all three domains of life. However, the understanding of DNA methylation in insects is limited. Here, we amplified the full-length transcripts of the DNA methyltransferases Nlu-Dnmt1 and Nlu-Dnmt3, indicating that a complete DNA methylation toolkit exists in the brown planthopper, Nilaparvata lugens, a destructive pest in rice production. Nlu-DNMT1 and Nlu-DNMT3 had the conserved motifs and domains of the DNA methyltransferase family. Nlu-Dnmt1 and Nlu-Dnmt3 were highly expressed in the mated and gravid female adults but weakly expressed in larvae, male adults, and virgin female adults. Silencing Nlu-Dnmt1 and Nlu-Dnmt3 in gravid brachypterous female adults led to fewer offspring, suggesting that DNA methylation regulates female fecundity in insects.

A comprehensive view of the epigenetic landscape part I: DNA methylation, passive and active DNA demethylation pathways and histone variants.

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer.

Abstract 420: Tet family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma

Abstract Objective: DNA methylation is a fundamental epigenetic process that modulates gene expression. Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Although the base 5-methylcytosine (5mc) and the DNA methyltransferase (DNMT) family of enzymes that catalyze DNA methylation are well-studied, the mechanisms of DNA demethylation are poorly understood. The Ten-eleven translocation (Tet) family of enzymes (Tet1, Tet2, and Tet3) has been implicated in DNA demethylation. These enzymes possess the dioxygenase activity and convert 5-mC to 5-hydroxymethylcytosine (5-hmC). Frequent Tet mutational inactivation or reduction has been reported to associate with decreased 5-hmC levels in various types of myeloid leukemia and melanoma. However, the status of 5hmC and Tet expression in esophageal squamous cell carcinoma (ESCC) remains still unknown. Method: We analyzed the 5-hmC status in DNA from ESCC frozen tissues utilizing the ELISA test, and 5-hmC expression was evaluated by immunohistochemistry. We next examined the expression levels of Tet family mRNA in both normal and tumor tissues by RT-qPCR. Results: 5-hmC level was significantly lower in ESCC than in paired normal tissue in ELISA test (n=33, P<0.0001). The similar result was observed in immunohistochemistry. We found that the Tet2 mRNA expression was significantly lower in ESCCs than paired normal tissues (n=32, P<0.0001). The expression level of Tet2 mRNA was significantly associated with the 5hmC status (n=31, r=0.54, P=0.0085). Finally, 5-hmC status did not correlate with overall survival (P = 0.65). Conclusion: The 5hmC expression was decreased in ESCC tissues, and was associated with Tet2 expression level, suggesting that 5hmC and Tet2 might play a crucial role in the development of ESCCs. Citation Format: Asuka Murata, Yoshifumi Baba, Ryuichi Karashima, Satoshi Ida, Yu Imamura, Takatsugu Ishimoto, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Tet family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2014-420

DNA methyltransferases 3a and 3b are differentially expressed in the early stages of a rat liver carcinogenesis model.

Carcinogenesis is driven by the accumulation of mutations and abnormal DNA methylation patterns, particularly the hypermethylation of tumor‑suppressor genes. Changes in genomic DNA methylation patterns are established by the DNA methyltransferases (DNMTs) family: DNMT1, DNMT3a and DNMT3b. The DNMTs are known to be overexpressed in tumors. However, when the DNMTs expression profile is altered in earlier stages of carcinogenesis remains to be elucidated. The resistant hepatocyte model (RHM) allows the analysis of the hepatocellular carcinoma (HCC) from the formation of altered cell foci to the appearance of tumors in rats. To investigate the DNMTs expression in this model, we first observed that timp3, rassf1a and p16 genes became methylated during cancer development by methylation‑specific PCR (MSP) and the bisulphate sequencing PCR (BSP) of timp3. The differential expression at the RNA and protein level of the three DNMTs was also assessed. dnmt1 expression was higher in tumors than in normal and early cancer stages. However, no evident overexpression of the enzyme was identified by immunohistochemistry. By contrast, DNMT3a and DNMT3b were consistently subexpressed in tumors. In the present study, we report a carcinogenesis model that does not feature the overexpression of DNMT1 but exhibits a transient expression of DNMT3a and DNMT3b.

Decreased DNA Methylations at the Progesterone Receptor Promoter A Induce Functional Progesterone Withdrawal in Human Parturition

The functional interaction of progesterone receptor (PR) isoforms PRA and PRB regulates myometrial transition from the resting state to excitation-contraction to initiate parturition. However, the regulatory mechanisms responsible for maintenance and functional alteration of the PRA and PRB expression levels during human pregnancy and term labor, respectively, remain unknown. Therefore, this study was designed to investigate whether and how epigenetic DNA modifications, specifically methylations, at the PRs' promoter regions contribute to the differential expression of PRA and PRB in laboring term myometrium of humans. Comparative analysis of PRA and PRB messenger RNA (mRNA) expression levels and accompanying changes in their promoters' methylation status was carried out using human myometrial samples from women undergoing singleton, term deliveries by cesarean section, either in the absence of labor (designated as NIL for not-in-labor) or in active labor (designated as IL for in labor). The PRA gene expression was shown to be elevated significantly during labor, while PRB gene expression was unaltered, and this differential expression was accompanied by decreased DNA methylation at the PRA promoter and not at the PRB promoter. In addition, labor-related decreased mRNA expression of the DNA methyltransferase (DNMT) family members DNMT1 and DNMT3a was found, however whether the increased expression of DNMTs directly supports the functional withdrawal of progesterone needs further investigation. Collectively, these data indicate that DNA methylation might represent an important epigenetic mechanism of labor-related differential expression of PRs, thereby mediating the biological process of functional PR withdrawal at term for parturition.

DNA methyctransferase 1 and gynecological malignancies

DNMT1,the main member of the DNA methyltransferase (DNMT) family,plays a core role in maintaining and regulating tumor cell genome-wide and the local area methylation.DNMT1 is highly expressed in gynecological malignancies,which leads to abnormal DNA methylation of genes,especially high methylation of tumor suppressor genes,gene expression silencing,and malignant cell growth. Key words: Site-specific DNA methyltransferase (cytosine-specific); Genital neoplasms,female

Characterization of DNA Methylation Variations During Fruit Development and Ripening of Sweet Orange

DNA methylation is a type of epigenetic modification involved in many biological processes. Recent studies have shown that DNA methylation plays important roles in regulating fruit development and ripening. In the present study, sweet oranges at six developmental stages were investigated to understand the possible roles of DNA methylation during fruit development and ripening. Analysis of genomic DNA methylation levels in different fruit tissues and developmental stages indicated that methylation levels were not static during fruit development and ripening. To explore the possible contributions of methylation-related genes in fruit development and ripening process, the full-length coding sequences corresponding to three types of DNA methyltransferase families (CsMET1, CsCMT3, and CsDRM1), a chromatin-remodeling gene (CsDDM1), and a demethylation gene (CsDME1) were isolated. The expression profiles of these methylation-related genes were analyzed in vegetative tissues (seedlings and leaves) and reproductive tissues (three stages of flowering and six stages of fruit development). The results showed that CsDRM1 was highly expressed in seedlings, leaves, and flowers compared with other genes. During fruit development and ripening, CsMET1, CsCMT1, CsCMT2, CsDRM1, and CsDMEs showed higher expression in the peel than in the flesh. However, global DNA methylation level changes could not be explained by the expression of a single gene, suggesting that the methylation and demethylation system may cooperatively contribute to the final global DNA methylation pattern.

The CpG island encompassing the promoter and first exon of human DNMT3L gene is a PcG/TrX response element (PRE).

DNMT3L, a member of DNA methyltransferases family, is present only in mammals. As it provides specificity to the action of de novo methyltransferases, DNMT3A and DNMT3B and interacts with histone H3, DNMT3L has been invoked as the molecule that can read the histone code and translate it into DNA methylation. It plays an important role in the initiation of genomic imprints during gametogenesis and in nuclear reprogramming. With important functions attributed to it, it is imperative that the DNMT3L expression is tightly controlled. Previously, we had identified a CpG island within the human DNMT3L promoter and first exon that showed loss of DNA methylation in cancer samples. Here we show that this Differentially Methylated CpG island within DNMT3L (DNMT3L DMC) acts to repress transcription, is a Polycomb/Trithorax Response Element (PRE) and interacts with both PRC1 and PRC2 Polycomb repressive complexes. In addition, it adopts inactive chromatin conformation and is associated with other inactive chromatin-specific proteins like SUV39H1 and HP1. The presence of DNMT3L DMC also influences the adjacent promoter to adopt repressive histone post-translational modifications. Due to its association with multiple layers of repressive epigenetic modifications, we believe that PRE within the DNMT3L DMC is responsible for the tight regulation of DNMT3L expression and the aberrant epigenetic modifications of this region leading to DNMT3L overexpression could be the reason of nuclear programming during carcinogenesis.

Dnmt1-Independent CG Methylation Contributes to Nucleosome Positioning in Diverse Eukaryotes

Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Comprehensive gene expression analysis of the DNA (cytosine-5) methyltransferase family in rice (Oryza sativa L.).

Cytosine DNA methylation is a conserved epigenetic regulatory mechanism in both plants and animals. DNA methyltransferases (DNA MTases) not only initiate (de novo) but also maintain the process of DNA methylation. Here, we characterized the genome-wide expression profiles of 10 cytosine DNA MTase genes belonging to 4 subfamilies, MET1, CMT, DNMT2, and DRM, in rice. Tissue-specific gene expression analysis showed that all family members varied widely in their expression and specificities and might be involved in some basic metabolic pathways. Similarly, the expression of all rice cytosine DNA MTase genes was not regulated by plant hormones except OsDRM1a and OsDRM1b, which were downregulated by jasmonic acid. The transcription level of 10 genes in rice shoots and roots was also measured under salt and osmotic stress. Meanwhile, quantitative polymerase chain reaction data of the japonica and indica rice cultivars revealed that there is large variation in the expression activities of all genes. The results provide a foundation to further explore the roles of DNA MTases and the epigenetic regulation of abiotic stress responses in rice.

Developmental expression of Arabidopsis methyltransferase genes MET1, DRM2 and CMT3

Cytosine methylation is an epigenetic mark found in the genome of fungi, plants, and animals. DNA methylation is catalyzed by DNA methyltransferases. The function of DNA methyltransferases was shown to be highly conversed, but biological role of these enzymes has not been clearly defined. We generated transgenic plants expressing METHYLTRANSFERASES::GUS reporter genes for three major DNA methyltransferases (MET1, DRM2 and CMT3) to gain insight into the potential physiological relevance of the distinct members of the DNA methyltransferase family in Arabidopsis thaliana, and to investigate the expression patterns in detail. We found that METHYLTRANSFERASE::GUS genes display unique tissue, cell-type, and temporal patterns of expression throughout normal development, particularly in the flower. Our findings are supported by semi-quantitative reverse-transcription PCR, as well as by analyses of microarray databases. These data suggest that DNA methyltransferase may contribute to morphogenesis at every developmental stage and in every plant organ.

Influence of environmental factors on DNA methylation

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs) that transfer a methyl group from S-adenyl methionine (SAM) to the fifth carbon of a cytosine residue to form 5mC. DNA methylation affects the interaction between the histone and DNA, which changes the chromosome structure and has reverse relationship with gene expression in general. Up to now, more and more studies have confirmed that environmental factors can alter epigenetic modifications, which do not involve in changing DNA sequence. So it can explain the phenotype of creature in a certain degree. This article focused on the influence of environmental factors, such as temperature, nutrient supply, heavy metal, early stress and radiation, on DNA methylation change. As a matter of fact, it does not only change the DNA methylation in parents and offspring but also their behavior and phenotype. Overall, this review will help us get better understanding of the relationship between environmental factors and gene expression regulation.

Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells

BackgroundHypermethylation of the promoter of the tumor suppressor gene RASSF1A silences its expression and has been found to be associated with advanced grade prostatic tumors. The DNA methyltransferase (DNMT) family of enzymes are known to be involved in the epigenetic silencing of gene expression, including RASSF1A, and are often overexpressed in prostate cancer. The present study demonstrates how mahanine, a plant-derived carbazole alkaloid, restores RASSF1A expression by down-regulating specific members of the DNMT family of proteins in prostate cancer cells.ResultsUsing methylation-specific PCR we establish that mahanine restores the expression of RASSF1A by inducing the demethylation of its promoter in prostate cancer cells. Furthermore, we show that mahanine treatment induces the degradation of DNMT1 and DNMT3B, but not DNMT3A, via the ubiquitin-proteasome pathway; an effect which is rescued in the presence of a proteasome inhibitor, MG132. The inactivation of Akt by wortmannin, a PI3K inhibitor, results in a similar down-regulation in the levels DNMT1 and DNMT3B. Mahanine treatment results in a decline in phospho-Akt levels and a disruption in the interaction of Akt with DNMT1 and DNMT3B. Conversely, the exogenous expression of constitutively active Akt inhibits the ability of mahanine to down-regulate these DNMTs, suggesting that the degradation of DNMT1 and DNMT3B by mahanine occurs via Akt inactivation.ConclusionsTaken together, we show that mahanine treatment induces the proteasomal degradation of DNMT1 and DNMT3B via the inactivation of Akt, which facilitates the demethylation of the RASSF1A promoter and restores its expression in prostate cancer cells. Therefore, mahanine could be a potential therapeutic agent for advanced prostate cancer in men when RASSF1A expression is silenced.

From paramutation to paradigm.

From paramutation to paradigm.

Abstract 3143: A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome.

Abstract Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally expressed during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors may differ biologically and have different prognosis in HCC patients. Using microarray-based global microRNA and mRNA profiling, we found that members of the miR-29 family are the most significantly (p<1E-5) down-regulated miRs in AFP+ tumors (n=242). Physiologically, during mouse embryonic development, miR-29 family expression is low but gradually increases after birth, contrary to AFP expression which dramatically decreases after birth. In addition, a member of the DNA methyltransferase (DNMT) family, DNMT3A, is one of the most significantly (p=8E-6) up-regulated genes in AFP+ HCC. Interestingly, there is a significant inverse correlation (p<1E-4) between miR-29 and DNMT gene expression, suggesting that they may be functionally antagonistic, which is consistent with the finding showing that DNMT3A and DNMT3B are direct targets of miR-29. Experimentally, we found that increased AFP expression or the addition of AFP+ media to AFP- cells inhibits miR-29a expression in HCC cell lines. We also found that AFP transcriptionally regulates the promoter of miR-29a. Moreover, global DNA methylation array profiling reveals increased methylation in HCC patients with high levels of serum AFP. These results support our hypothesis that AFP inhibits miR-29, which modulates epigenetic changes that contribute to poor outcome. Taken together, this study elucidates the mechanisms that link AFP and miR-29 expression to epigenetic alterations and may aid in the development of novel therapeutic agents to improve survival of HCC patients. Citation Format: Sonya Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Christopher Loffredo, Xin Wei Wang. A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2013-3143