Genes Encoding DNA Methyltransferases Research Articles

A second hit by PFOS exposure exacerbated developmental defects in medaka embryos with a history of ancestral BPA exposure

Bisphenol-A (BPA), a known endocrine-disrupting chemical (EDC) in plastics and resins, has been found to induce heritable health effects in fish and mammals, affecting directly exposed individuals and indirectly their progenies in subsequent generations. It is not clearly understood if subsequent generations of the BPA-exposed ancestors have increased sensitivity to the second hit by the chemicals of emerging concern. To understand this, the present study examined the effects of developmental exposure to perfluorooctanesulfonic acid (PFOS), which has been a global contaminant recently, in embryos whose ancestors were exposed to BPA. Two lineages of medaka (Oryzias latipes) were established: 1) the BPA lineage in which the F0 generation was exposed to 10 μg/L BPA during early development and 2) the control lineage with no BPA exposure in the F0 generation. These lineages were raised up to the F4 generation without further exposure. The embryos of the F4 generation were exposed to PFOS at 0, 0.002, 0.02, 0.2, 2, and 20 mg/L concentrations. Early developmental defects resulting in mortality, delayed hatching, teratogenic phenotypes, and altered gene expression were examined in both lineages. The expression level of genes encoding DNA methyltransferases and genes responsible for oxidative stress defense were determined. Following environmentally relevant PFOS exposure, organisms with a history of BPA exposure displayed significant changes in all categories of developmental defects mentioned above, including increased expression of genes related to oxidative stress, compared to individuals without BPA exposure. The present study provides initial evidence that a history of ancestral BPA exposure can alter sensitivity to developmental disorders following the second hit by PFOS exposure. The variable of ancestral BPA exposure could be considered in mechanistic, medical, and regulatory toxicology, and can also be applied to holistic environmental equity research.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Транскрипционная активность генов ДНК-метилтрансфераз у жителей Уральского региона, подвергшихся хроническому радиационному воздействию

In addition to damaging the genetic apparatus of the cell, ionizing radiation can cause epigenetic alterations. DNA methylation that plays a vital part in regulation of cellular processes is a common epigenetic modification. DNA methylation ensured by DNA methyltransferases occurs in the CpG-rich sequences. The study was aimed to assess mRNA expression of genes encoding DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) in the chronically exposed individuals who live along the River Techa over a long-term period. A total of 112 people were examined more than 65 years after the beginning of chronic exposure. The average accumulated dose to red bone marrow (RBM) was 782.0 ± 82.3 mGy, and the average accumulated dose to thymus and peripheral lymphoid organs was 93.2 ± 13.6 mGy. The subjects' age at the time of examination was 67.9 ± 0.8 years (54–83 years). The relative mRNA levels for the studied genes were assessed by realtime polymerase chain reaction (real-time PCR). mRNA expression of DNMT1 correlated positively with the dose to RBM (p = 0.04), thymus and peripheral lymphoid organs (p = 0.02), as well as with the dose rate in these organs (p = 0.05, p = 0.04, respectively) during the period of the highest levels of radiation exposure. In individuals exposed in the high dose range (over 1000 mGy) there was a significant increase in the expression of DNMT1 mRNA compared to the comparison group (p = 0.02). The findings may indicate the DNMT1 gene involvement in epigenetic alterations that occur in the chronically exposed people in the long term.

Evaluation of the initial manifestations of the toxic process in conditions of chronic action of low subtoxic doses of dioxins polluting the environment

AIM: To study the state of the genome by indicators of retrotransposon activity the gene encoding DNA methyltransferase 1 (DNMT1); DNA damage in animals from the natural population of the bank vole (Clethrionomys glareolus) living in the vicinity of a preserved landfill contaminated with low concentrations of dioxins (landfill of production and consumption waste "Salariyevo", Moscow).
 MATERIAL AND METHODS: The activity of ERV-L, B1, and L1 retrotransposons and the transcription level of the DNMT1 gene were evaluated by real-time PCR. The stability of DNA in liver and bone marrow cells was characterized by the comet assay method. Afterward, the obtained characteristics of the state (stability, reactivity, and damage) of the genome in response to environmental stress factors were compared in groups of animals from the study and conditionally control samples.
 RESULTS: The effects of a decrease in the activity of retrotransposons of classes B1 and L1, and an increase in DNMT1 gene expression level were revealed in voles from the natural population living under the long-term chronic exposure to low doses of dioxins decrease in the activity of retrotransposons of classes B1 and L1, and an increase in the expression level of the DNMT1 gene were revealed. An increased level of DNA damage (on average up to 56% of the DNA in the tail of the comet) was detected in hepatocytes, with the addition of winter environmental factors to the chronic effect of small subtoxic doses of dioxins.
 CONCLUSION: Suppression of retrotransposon activity and increased expression of its epigenetic regulator (DNMT1) are regarded as adaptive responses to long-term chronic exposure to low doses of dioxins polluting the environment. The alteration in the reactivity and destabilization of the genome indicates the launch of the initial mechanisms of the toxic process formation. The created and tested methodological base for its study opens up prospects for establishing a threshold level, and as a result, substantiating indicators for screening assessment of local (territorial) risk to public health by biomonitoring.

Somatic Dnmt3a inactivation leads to slow, canonical DNA methylation loss in murine hematopoietic cells

SummaryMutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis and are among the most common initiating events of acute myeloid leukemia (AML). Studies in germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells; however, the kinetics of methylation loss following acquired DNMT3A inactivation in hematopoietic cells is essentially unknown. Therefore, we evaluated a somatic, inducible model of hematopoietic Dnmt3a loss, and show that inactivation of Dnmt3a in murine hematopoietic cells results in a relatively slow loss of methylation at canonical sites throughout the genome; in contrast, remethylation of Dnmt3a deficient genomes in hematopoietic cells occurs much more quickly. This data suggests that slow methylation loss may contribute, at least in part, to the long latent period that characterizes clonal expansion and leukemia development in individuals with acquired DNMT3A mutations in hematopoietic stem cells.

Dnmt3a Inactivation Leads to Slow DNA Methylation Loss in Murine Hematopoietic Cells In Vivo

IntroductionSomatic mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis, and among the most common initiating events for acute myeloid leukemia (AML). Constitutional DNMT3A mutations cause DNMT3A Overgrowth Syndrome, associated with obesity, overgrowth, behavioral problems, and increased risk of hematopoietic malignancies after years of latency [1]. Studies of germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells. This hypomethylation phenotype is thought to contribute to the epigenetic “fitness” of hematopoietic cells to acquire cooperating mutations that cause clonal progression and AML. However, the kinetics of methylation loss following mutational inactivation of DNMT3A in hematopoietic cells is essentially unknown.MethodsBone marrow from C57Bl/6-CD45.2 Dnmt3a fl/fl -ERT2-cre mice was transplanted into lethally irradiated C57Bl/6-CD45.1 recipients. Engraftment frequencies of >80% were detected in virtually all mice. Inactivation of Dnmt3a was induced with 9 doses of Tamoxifen (tam) given by gavage on MWF for three consecutive weeks. Bone marrow samples were serially collected for whole-genome bisulfite sequencing (WGBS) and analysis as previously described [1-4]. We compared these data to WGBS data from previously published doxycycline inducible DNMT3A transgene addback data, described in [3].ResultsCre-mediated deletion of exon 19 (flanked by loxP sites) leads to out-of-frame splicing of the mRNAs encoding all active isoforms of Dnmt3a. To limit Dnmt3a inactivation to hematopoietic cells, we used a transplantation model, which allowed us to eliminate the effects Dnmt3a loss in non-hematopoietic tissues. Floxing efficiency in the bone marrow was >90% 1 week after the last dose of Tam, and these levels persisted until the last mice in the cohort were analyzed 35 weeks later. We defined differentially methylated regions (DMRs) using WGBS data derived from WT vs Dnmt3a knockout (KO) bone marrow samples, using previously described criteria [4]. We identified 33,519 focal, highly canonical DMRs in the KO samples; 33,504 (99.96%) of these were hypomethylated, compared to WT marrow. Passively plotting the methylation values of the Dnmt3afl/fl + tam (“takeaway”) samples for these DMRs over time (Figure 1a) revealed a slow, progressive, ordered loss of methylation, although the degree of methylation loss did not reach KO levels by 36 weeks. In contrast, remethylation of most of these DMRs with transgene-driven DNMT3A “addback” in KOmice revealed methylation ‘correction’ that started rapidly (within 1-2 weeks), and that approached wild-type levels by 24 weeks (Figure 1b). Combined, these data show that methylation loss after Dnmt3a inactivation occurs much more slowly than remethylation of Dnmt3a-dependent sites following restoration of DNMT3A activity. This trend was true across all functionally annotated regions of the genome, including gene bodies, promoters, enhancers, transcriptional start sites (TSS) and CpG islands. Importantly, DMRs that lost methylation rapidly in the takeaway setting, also gained methylation rapidly with addback, suggesting that there is a canonical set of genomic regions where DNA methylation is actively “remodeled”. These “Dynamic DMRs” are significantly smaller (and had fewer CpGs) than DMRs associated with slower rates of remodeling.ConclusionsThese data describe a very slow rate of methylation loss following Dnmt3a inactivation in hematopoietic cells. We also describe regions of the genome (Dynamic DMRs) where DNA methylation is actively remodeled in hematopoietic cells, presumably by the collaborative action of de novo DNA methylases (Dnmt3a and Dnmt3b) vs. the DNA demethylases (i.e. the TET family of enzymes). Since clonal hematopoiesis patients with DNMT3A mutations can have very long latent periods before developing hematopoietic malignancies, these data suggest that long latency may be (at least in part) related to the very slow loss of DNA methylation at relevant regions of the genome after a DNMT3A allele acquires an inactivating somatic mutation. [Display omitted] DisclosuresLeight: Amgen: Current Employment.

CBFB-MYH11 Fusion Sequesters RUNX1 in Cytoplasm to Prevent DNMT3A Recruitment to Target Genes in AML.

A growing number of human diseases have been found to be associated with aberrant DNA methylation, including cancer. Mutations targeting genes encoding DNA methyltransferase (DNMT), TET family of DNA demethylases, and isocitrate dehydrogenase (IDH1, IDH2) that produce TET inhibitory metabolite, 2-hyoxyglutarate (2-HG), are found in more than half of acute myeloid leukemia (AML). To gain new insights into the regulation of DNA de/methylation and consequence of its alteration in cancer development, we searched for genes which are mutated in a manner that is linked with gene mutations involved in DNA de/methylation in multiple cancer types. We found that recurrent CBFB-MYH11 fusions, which result in the expression of fusion protein comprising core-binding factor β (CBFB) and myosin heavy chain 11 (MYH11) and are found in 6∼8% of AML patients, occur mutually exclusively with DNMT3A mutations. Tumors bearing CBFB-MYH11 fusion show DNA hypomethylation patterns similar to those with loss-of-function mutation of DNMT3A. Expression of CBFB-MYH11 fusion or inhibition of DNMT3A similarly impairs the methylation and expression of target genes of Runt related transcription factor 1 (RUNX1), a functional partner of CBFB. We demonstrate that RUNX1 directly interacts with DNMT3A and that CBFB-MYH11 fusion protein sequesters RUNX1 in the cytoplasm, thereby preventing RUNX1 from interacting with and recruiting DNMT3A to its target genes. Our results identify a novel regulation of DNA methylation and provide a molecular basis how CBFB-MYH11 fusion contributes to leukemogenesis.

Dnmt3a-null hematopoietic stem and progenitor cells expand after busulfan treatment

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.

Virus-mediated Dnmt1 and Dnmt3a deletion disrupts excitatory synaptogenesis and synaptic function in primary cultured hippocampal neurons

Dnmt1, Dnmt3a and Dnmt3b are main genes encoding DNA methyltransferases (Dnmts) which catalyze DNA methylation and regulate gene expression without changing DNA sequence. Our previous study disclosed that double knockout of Dnmt1 and Dnmt3a in forebrain excitatory neurons impaired synaptic plasticity and led to hippocampus-dependent learning and memory deficits, however the underlying synaptic mechanisms remain uncertain. In this study, we selectively knocked down the expression of Dnmt1 and Dnmt3a in primary cultured hippocampal neurons derived from embryonic Dnmt1,3a2flox/2flox mice by transfection with Cre-expressing virus, to study the effect of Dnmts and mediated DNA methylation on synaptogenesis and synaptic function. We found that the hippocampal neurons at 15 days in vitro (DIV15) exhibited similar size of cell body, but longer dendrites with reduced number of branches and lower density of excitatory synapses formation after virus-mediated Dnmt1 and Dnmt3a deletion. Supportively, cultured neurons with Dnmt1 and Dnmt3a deficiency displayed reduced frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), indicating that both pre- and post-synaptic dysfunction are involved. In addition, our Ca2+-image study with Rhod-3AM revealed suppression of glutamate-evoked elevation of cytoplasmic [Ca2+] after Dnmt1 and Dnmt3a deletion. Altogether our findings provide new evidence that normal expression of Dnmt1 and Dnmt3a in hippocampal neurons are essential for excitatory synaptogenesis and synaptic function.

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice

DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-β signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.

Identification and sequencing of the gene encoding DNA methyltransferase 3 (DNMT3) from sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces saponins, and has a high potential to cope with environmental stress, such as aestivation, organ regeneration, and wound healing. Recently, several studies have shown that cellular reprogramming and the physiological responses of the sea cucumber to environmental changes, including aestivation, are potentially mediated by epigenetic DNA methylation. The DNA methyltransferase (DNMT)1 and DNMT3 genes are independent participants in the maintenance and de novo methylation of specific sequences. Sea urchin (Strongylocentrotus purpuratus) and starfish (Asterina pectinifera), which belong to the same phylum as A. japonicus, have both DNMT1 and DNMT3 genes. However, it was previously reported that DNMT1 is present, but DNMT3 is absent, in A. japonicus. In the present study, we sequenced the full-length cDNA of the A. japonicus DNMT3 gene. The newly sequenced DNMT3 gene comprises three major conserved domains (Pro-Trp-Trp-Pro (PWWP), plant homeodomain (PHD), and S-adenosylmethionine-dependent methyltransferase (AdoMet-MTase)), indicating that the DNMT3 possibly has de novo DNA methylation catalytic activity. Gene structure and phylogenetic analysis showed that sea cucumber DNMT3 is evolutionarily conserved in the Echinodermata. Next, we demonstrated the conservation of DNMT3 gene expression in sea cucumber and starfish belong to same phylum, echinoderm. Using reverse transcription-polymerase chain reaction, sea cucumber DNMT3 mRNA was detected in testis tissue, but not in other tissues tested, including the respiratory tree, muscle, tentacle, intestine, and ovary. This is inconsistent with previous reports, which showed the expression of DNMT3 in ovary, but not in testis of the starfish A. pectinifera, indicating the tissue- and species-specific expression of DNMT3 gene. Although further studies are needed to clarify the epigenetic regulatory mechanisms of DNMT3 and its application to the aquaculture industry, our findings may provide insights into the sea cucumber biology.

Examining multi- and transgenerational behavioral and molecular alterations resulting from parental exposure to an environmental PCB and PBDE mixture

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants extensively used during the 20th century and still present in aquatic environments despite their ban. Effects of exposure to these compounds over generations are poorly documented. Therefore, our aims were to characterize behavioral responses and underlying molecular mechanisms in zebrafish exposed to an environmentally relevant mixture of PCBs and PBDEs as well as in four unexposed offspring generations. Zebrafish (F0) were chronically exposed from the first meal onward to a diet spiked with a mixture containing 22 PCB and 7 PBDE congeners in proportions and concentrations reflecting environmental situations (ΣPCBs = 1991 and ΣPBDEs = 411 ng/g). Four offspring generations (F1 to F4) were obtained from this F0 and were not further exposed. Behavior was assessed at both larval and adult stages. Mechanisms related to behavioral defects (habenula maturation and c-fos transcription) and methylation (dnmts transcription) were monitored in larvae. Exposed adult F0 as well as F1 and F3 adults displayed no behavioral change while F2 expressed anxiety-like behavior. Larval behavior was also disrupted, i.e. hyperactive after light to dark transition in F1 or hypoactive in F2, F3 and F4. Behavioral disruptions may be related to defect in habenula maturation (observed in F1) and change in c-fos transcription (observed in F1 and F2). Transcription of the gene encoding DNA methyltransferase (dnmt3ba) was also modified in all generations. Our results lead us to hypothesize that chronic dietary exposure to an environmentally relevant mixture of PCB and PBDE triggers multigenerational and transgenerational molecular and behavioral disruptions in a vertebrate model.

Mutation of the DNMT3A and IDH1/2 genes in Iranian acute myeloid leukemia patients with normal karyotype (CN-AML): association with other gene mutation and clinical and laboratory characteristics

Mutation of the genes encoding DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1/2 (IDH 1/2) is among the most commonly occurring mutation found in acute myeloid leukemia (AML) patients. This study was purposed to investigate the frequency of DNMT3A and IDH1/2 gene mutation and the clinical features of Iranian cytogenetically normal acute myeloid leukemia (CN-AML) patients harboring these mutations. Thirty-nine CN-AML patients were recruited at the time of diagnosis. PCR followed by direct sequencing was used to detect the mutations of DNMT3A (R882), IDH1 (R132), and IDH2 (R140 and R172). The results showed that of all CN-AML patients, DNMT3A, IDH1, and IDH2 mutations were observed in five (12.8%), five (12.8%), and five (13.2%) patients, respectively. In addition, the most frequent DNMT3A, IDH1, and IDH2 mutant types were R882C, R132C, and R140Q types, respectively. Our results also described that both DNMT3A and IDH1/2 mutations were not associated with significant change in hemoglobin (Hb) levels, white blood cell (WBC) and platelet count, and bone marrow blast percentage (P > 0.05). There was also no significant difference in the mutation status of DNMT3A and IDH1/2 genes regarding age and gender (P > 0.05). A positive relationship was observed between the co-occurrence of DNMT3A and IDH2 (P = 0.021) and FLT3-ITD and NPM1 (P = 0.044). Increasing sample size and longer follow-up of patients may provide additional data to understand the prognostic significance of the DNMT3A and IDH1/2 mutation in the management of CN-AML patients.

Transgenerational effects of prenatal restricted diet on gene expression and histone modifications in the rat

Dietary triggers acting on a developing fetus can affect the functioning of the body in later life; this can be observed on various levels, including epigenetic modifications and gene expression. Early-life programmed changes may be transmitted to successive generations. In this study, the impact of prenatal restricted diet was studied in four generations of rats. We hypothesized that this diet can induce changes in the expression of major genes involved in two epigenetic mechanisms: DNA methylation and histone modifications. The transcript level of six genes involved in these processes (Dnmt1, Dnmt3a, Dnmt3b, Mecp2, Hdac1, and Sin3a) was therefore determined in three tissues (liver, adipose, and muscle). This diet was found to have no effect on the F0 pregnant females. In the F1 progeny (fetuses at day 19 of pregnancy and 4-week-old rats) significant differences in the expression of the genes were observed mostly in the liver; in subsequent generations, we therefore studied only this tissue. Among the genes encoding DNA methyltransferases, significant changes were observed for Dnmt1 in the F1 animals from the restricted group, but these were no longer evident in F2 and F3. The Dnmt3a and Dnmt3b genes showed no differences in mRNA level in F1 fetuses. Concerning the transcript level of the Mecp2 gene only in F1 generation significant changes were found. For the histone modification genes, an increase in the expression of Hdac1 in fetus liver was found in F1 and F2, while its level decreased in F3. The abundance of the Sin3a transcript varied in all generations. It was also found that the mRNA levels of the studied genes correlated highly positive with each other, but only in fetuses from the F1 restricted group. The DNA methylation cell potential, defined as the ratio of SAM (S-adenosylmethionine) to SAH (S-adenosylhomocysteine), was measured in the liver, with no alterations being found in the restricted groups. Evaluation of global histone H3 acetylation showed that it underwent a significant increase in the fetal livers of F1, while during aging (four-week old animals) this difference was no longer maintained. A tendency of increased H3 acetylation in fetuses was also detected in F2 generation. In F1 fetuses from restricted group the increased H3 acetylation positively correlated with transcriptional status of the studied genes. Our results indicate that the prenatal restriction diet can affect the activity of genes involved in epigenetic mechanisms in the liver across generations. Moreover, this feeding type influenced the global histone H3 acetylation in fetal liver.

A comparative analysis of DNA methylation in diploid and tetraploid apple (Malus × domestica Borkh.)

DNA methylation is one of the major epigenetic modifications. It is very important to the regulation of gene expression. Methylation-sensitive amplification polymorphism (MSAP) profiling was applied to a diploid apple cultivar and its derived autotetraploid in order to characterize the level and pattern of DNA methylation at the two different ploidies. The frequency of methylated restriction sites was very similar between the two types (28.0% vs 27.3%), implying that polyploidization had a low effect on the global level of DNA methylation. However, with respect to the pattern of methylation, the frequency of hemi-methylated sites was higher in the tetraploid. When the transcription level of three genes encoding DNA methyltransferase was investigated in various tissues, it was established that MET1 transcript abundance was the lowest of the three genes throughout the plant, while that of DRM2 was high in the leaf, flower and fruit, as was that of CTM3 in the fruit. Polyploidization had no discernible effect on the transcription level of any of the three genes.

Epigenetic and genetic alterations and their influence on gene regulation in chronic lymphocytic leukemia

BackgroundTo understand the changes of gene regulation in carcinogenesis, we explored signals of DNA methylation – a stable epigenetic mark of gene regulatory elements — and designed a computational model to profile loss and gain of regulatory elements (REs) during carcinogenesis. We also utilized sequencing data to analyze the allele frequency of single nucleotide polymorphisms (SNPs) and detected the cancer-associated SNPs, i.e., the SNPs displaying the significant allele frequency difference between cancer and normal samples.ResultsAfter applying this model to chronic lymphocytic leukemia (CLL) data, we identified REs differentially activated (dREs) between normal and CLL cells, consisting of 6,802 dREs gained and 4,606 dREs lost in CLL. The identified regulatory perturbations coincide with changes in the expression of target genes. In particular, the genes encoding DNA methyltransferases harbor multiple lost-in-cancer dREs and zero gained-in-cancer dREs, indicating that the damaged regulation of these genes might be one of the key causes of tumor formation. dREs display a significantly elevated density of the genome-wide association study (GWAS) SNPs associated with CLL and CLL-related traits. We observed that most of dRE GWAS SNPs associated with CLL and CLL-related traits (83%) display a significant haplotype association among the identified cancer-associated alleles and the risk alleles that have been reported in GWAS. Also dREs are enriched for the binding sites of the well-established B-cell and CLL transcription factors (TFs) NF-kB, AP2, P53, E2F1, PAX5, and SP1. We also identified CLL-associated SNPs and demonstrated that the mutations at these SNPs change the binding sites of key TFs much more frequently than expected.ConclusionsThrough exploring sequencing data measuring DNA methylation, we identified the epigenetic alterations (more specifically, DNA methylation) and genetic mutations along non-coding genomic regions CLL, and demonstrated that these changes play a critical role in carcinogenesis through damaging the regulation of key genes and alternating the binding of key TFs in B and CLL cells.

Concerted changes in transcriptional regulation of genes involved in DNA methylation, demethylation, and folate-mediated one-carbon metabolism pathways in the NCI-60 cancer cell line panel in response to cancer drug treatment.

BackgroundAberrant patterns of DNA methylation are abundant in cancer, and epigenetic pathways are increasingly being targeted in cancer drug treatment. Genetic components of the folate-mediated one-carbon metabolism pathway can affect DNA methylation and other vital cell functions, including DNA synthesis, amino acid biosynthesis, and cell growth.ResultsWe used a bioinformatics tool, the Transcriptional Pharmacology Workbench, to analyze temporal changes in gene expression among epigenetic regulators of DNA methylation and demethylation, and one-carbon metabolism genes in response to cancer drug treatment. We analyzed gene expression information from the NCI-60 cancer cell line panel after treatment with five antitumor agents, 5-azacytidine, doxorubicin, vorinostat, paclitaxel, and cisplatin. Each antitumor agent elicited concerted changes in gene expression of multiple pathway components across the cell lines. Expression changes of FOLR2, SMUG1, GART, GADD45A, MBD1, MTR, MTHFD1, and CTH were significantly correlated with chemosensitivity to some of the agents. Among many genes with concerted expression response to individual antitumor agents were genes encoding DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, epigenetic and DNA repair factors MGMT, GADD45A, and MBD1, and one-carbon metabolism pathway members MTHFD1, TYMS, DHFR, MTR, MAT2A, SLC19A1, ATIC, and GART.ConclusionsThese transcriptional changes are likely to influence vital cellular functions of DNA methylation and demethylation, cellular growth, DNA biosynthesis, and DNA repair, and some of them may contribute to cytotoxic and apoptotic action of the drugs. This concerted molecular response was observed in a time-dependent manner, which may provide future guidelines for temporal selection of genetic drug targets for combination drug therapy treatment regimens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0240-3) contains supplementary material, which is available to authorized users.

Phosphate starvation induces DNA methylation in the vicinity of cis-acting elements known to regulate the expression of phosphate–responsive genes

ABSTRACTPhosphate (Pi) limitation is a constraint for plant growth in many natural and agricultural ecosystems. Plants possess adaptive mechanisms that enable them to cope with conditions of limited Pi supply, including a highly regulated genetic program controlling the expression of genes involved in different metabolic, signaling and development processes of plants. Recently, we showed that in response to phosphate limitation Arabidopsis thaliana sets specific DNA methylation patterns of genic features that often correlated with changes in gene expression. Our findings included, dynamic methylation changes in response to phosphate starvation and the observation that the expression of genes encoding DNA methyltransferases appear to be directly controlled by the key regulator PHOSPHATE RESPONSE 1 (PHR1). These results provide insight into how epigenetic marks can influence plant genomes and transcriptional programs to respond and adapt to harsh conditions. Here we present an analysis of DNA methylation in the upstream regions of low Pi responsive genes in Arabidopsis seedlings exposed to low Pi conditions. We found that hypo- and hyper-methylation in the vicinity of cognate binding sites for transcription factors known to regulate the phosphate starvation response clearly correlates with increased or decreased expression of low-Pi responsive genes.

Genomic DNA Methylation Analyses Reveal the Distinct Profiles in Castor Bean Seeds with Persistent Endosperms.

Investigations of genomic DNA methylation in seeds have been restricted to a few model plants. The endosperm genomic DNA hypomethylation has been identified in angiosperm, but it is difficult to dissect the mechanism of how this hypomethylation is established and maintained because endosperm is ephemeral and disappears with seed development in most dicots. Castor bean (Ricinus communis), unlike Arabidopsis (Arabidopsis thaliana), endosperm is persistent throughout seed development, providing an excellent model in which to dissect the mechanism of endosperm genomic hypomethylation in dicots. We characterized the DNA methylation-related genes encoding DNA methyltransferases and demethylases and analyzed their expression profiles in different tissues. We examined genomic methylation including CG, CHG, and CHH contexts in endosperm and embryo tissues using bisulfite sequencing and revealed that the CHH methylation extent in endosperm and embryo was, unexpectedly, substantially higher than in previously studied plants, irrespective of the CHH percentage in their genomes. In particular, we found that the endosperm exhibited a global reduction in CG and CHG methylation extents relative to the embryo, markedly switching global gene expression. However, CHH methylation occurring in endosperm did not exhibit a significant reduction. Combining with the expression of 24-nucleotide small interfering RNAs (siRNAs) mapped within transposable element (TE) regions and genes involved in the RNA-directed DNA methylation pathway, we demonstrate that the 24-nucleotide siRNAs played a critical role in maintaining CHH methylation and repressing the activation of TEs in persistent endosperm development. This study discovered a novel genomic DNA methylation pattern and proposes the potential mechanism occurring in dicot seeds with persistent endosperm.

Plant DNA Methyltransferase Genes: Multiplicity, Expression, Methylation Patterns.

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the "body-methylated gene" prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.