Methylation Of Cytosine Residues Research Articles

Microsphere-Based Assessment of DNA Methylation for AML Prognosis.

Epigenetic dysregulation, including aberrant methylation of cytosine residues in DNA, is a hallmark of cancer and clearly results in oncogenic cellular alterations such as transcriptional attenuation of tumor suppressors and genomic instability. A number of studies have examined DNA methylation alterations in patients with acute myeloid leukemia (AML) and have shown that analysis of multilocus methylation patterns can identify biologically distinct AML subclasses and can predict patient prognosis. In order to utilize the prognostic capability of methylation analysis in a clinical setting, we have developed a microsphere-based HpaII tiny fragment enrichment by ligation-mediated PCR (xMELP) assay to interrogate the methylation state of genomic multiple loci along with a random forest-based classification algorithm that correlates DNA methylation status with patient prognosis. These tools can be easily implemented in a clinical molecular pathology laboratory and can be utilized for more accurate risk stratification of AML patients.

In Situ Analysis of DNA Methylation in Plants.

Epigenetic regulation in the plant genome is associated with the determination of expression patterns of various genes. Methylation of DNA at cytosine residues is one of the mechanisms of epigenetic regulation and has been a subject of various studies. Various techniques have been developed to analyze DNA methylation, most of which involve isolation of chromatin from cells and further in vitro studies. Limited techniques are available for in situ study of DNA methylation in plants. Here, we present such an in situ method for DNA methylation analysis which has high sensitivity and good reproducibility.

Tissue-specific mutation accumulation in human adult stem cells during life.

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases.

Methylation of DNA and histone proteins are mutually involved in the epigenetic regulation of gene expression mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). DNMTs methylate cytosine residues within gene promoters, whereas HMTs catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins, thus causing chromatin condensation and transcriptional repression, which play an important role in the pathogenesis of cancer. The potential reversibility of epigenetic alterations has encouraged the development of dual pharmacologic inhibitors of DNA and histone methylation as anticancer therapeutics. Dietary flavones can affect epigenetic modifications that accumulate over time and have shown anticancer properties, which are undefined. Through DNA binding and in silico protein-ligand docking studies with plant flavones viz. Apigenin, Chrysin and Luteolin, the effect of flavones on DNA and histone methylation was assessed. Spectroscopic analysis of flavones with calf-thymus DNA revealed intercalation as the dominant binding mode, with specific binding to a GC-rich sequence in the DNA duplex. A virtual screening approach using a model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones are tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Epigenetic studies performed with flavones exhibited a decrease in DNMT enzyme activity and a reversal of the hypermethylation of cytosine bases in the DNA and prevented cytosine methylation in the GC-rich promoter sequence incubated with the M.SssI enzyme. Furthermore, a marked decrease in HMT activity and a decrease in EZH2 protein expression and trimethylation of H3K27 were noted in histones isolated from cancer cells treated with plant flavones. Our results suggest that dietary flavones can alter DNMT and HMT activities and the methylation of DNA and histone proteins that regulate epigenetic modifications, thus providing a significant anticancer effect by altering epigenetic processes involved in the development of cancer.

Evaluation of DNMT1 gene expression profile and methylation of its promoter region in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is an autoimmune disease with a chronic inflammatory arthritis. The critical role of methylation in the biology of immunocytes has increasingly been surveyed to discover disease etiology. DNA methyltransferase 1 (DNMT1) is an enzyme, which establishes and regulates patterns of methylated cytosine residues. The aim of the current investigation was to unveil if methylation circumstances of CpG sites in DNMT1 promoter could affect the mRNA expression level of this gene in peripheral blood mononuclear cells (PBMCs) from AS patients. PBMCs were isolated from whole blood of 40 AS patients and 40 healthy individuals. Total RNA and DNA contents of leukocytes were extracted. Afterward, quantitative analysis was carried out by real-time PCR using the SYBR Green PCR Master Mix. Finally, to determine the methylation level, PCR products of bisulfite-treated DNA from patients and controls were sequenced. Compared with healthy controls, expression level of DNMT1 in AS patients was significantly downregulated. Methylation of DNMT1 promoter was significantly higher in AS patients in comparison to controls. While a negative correlation between methylation and expression level of DNMT1 was observed in AS patients, both methylation and expression level of DNMT1 did not correlate with clinical manifestations. Considering the observation that decreased expression level of DNMT1 was associated with hypermethylation of DNMT1 promoter in PBMCs from AS patients, this survey suggests that dysregulation of DNMT1 expression through altered methylation level of other target genes would probably contribute to AS development.

Abstract 5258: Inhibition of DNA methyltransferases and histone methyltransferases by plant flavones

Abstract Methylation of DNA and histone protein is an epigenetic modification mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs) critically involved in methylation and regulation of affinity binding between the histones and DNA backbone. HMTs catalyze the transfer of one, two, or three methyl groups to lysine and arginine residues of histone proteins. The HMTs-mediated increase in histone affinity to DNA causes chromatin condensation, preventing transcription; whereas DNMTs methylate cytosine residues within gene promoters resulting in transcriptional repression. DNMT and HMT activities are reported to be associated with signal transduction, cell growth and death, as well as with the pathogenesis of various human diseases including cancer. Dietary plant flavones can affect epigenetic modifications accumulated over time and have shown health-beneficial effects. However, the epigenetic response on DNMT and HMT elicited by plant flavones has not been elucidated. Through in silico protein-ligand docking studies and molecular studies with plant flavones viz. chrysin, apigenin and luteolin and their effect on DNA and histone methylation was assessed. The ligands were individually docked into the pocket of DNMT and EZH2 using Glide in XP (extra precision) mode (Schrodinger, LLC). Binding of ligands with proteins were evaluated using GlideScore, which is an empirically derived scoring function. Virtual screening approach using the model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Flavones exhibited a high docking rank (Glide score) in the order of chrysin (-5.8), apigenin (-6.4), luteolin (-7.4) compared with the pharmacological inhibitor, 5-Aza-2′-deoxycytidine (-4.2). Notably, all three flavones inhibited EZH2, having a high docking rank compared to the known pharmacological inhibitor, 3-Deazaneplanocin A (DZNep). The docking rank for chrysin was -10.07, apigenin -9.73 and luteolin -11.23, compared to -7.62 for DZNep. Epigenetic studies performed with plant flavones demonstrated reversal of hypermethylation of cytosine bases in the DNA and prevented methylation of cytosine in the GC-rich promoter sequence incubated with MSsI enzyme. Furthermore, decrease protein expression of EZH2 and trimethylation of H3K27 was noted in prostate cancer cells treated with these plant flavones. Taken together, our results suggest that plant flavone can alter DNMT and HMT activities and methylation of DNA and histone proteins that regulate epigenetic modifications providing significant health-effects and prevent various pathological processes involved in the development of cancer. Citation Format: Rajnee Kanwal, Manish Datt, Sanjay Gupta. Inhibition of DNA methyltransferases and histone methyltransferases by plant flavones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5258.

A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana

Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes.

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways.

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy.

Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin.

In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin. In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible.

DNA Methylation of BDNF Gene in Schizophrenia.

BackgroundAlthough genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia.Material/MethodsThe study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA.ResultsThere was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients.ConclusionsAlthough there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation.

Expression of the C-Terminal Domain of Mammalian &amp;lt;i&amp;gt;TET&amp;lt;/i&amp;gt;3 DNA Dioxygenase in &amp;lt;i&amp;gt;Arabidopsis thaliana&amp;lt;/i&amp;gt; Induces Heritable Methylation Changes at &amp;lt;i&amp;gt;rDNA&amp;lt;/i&amp;gt; Loci

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.

Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy.

Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM) at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p < 10−6), OR = 2.4; and p < 0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma

Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer.Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used.Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation.Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a.With support of the grant NT14393. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

5-Azacitidine induces demethylation of PTPL1 and inhibits growth in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) consists of various lymphoid malignancies with a diverse clinical pathology and biological characteristics. Methylation of cytosine residues by DNA methyltransferases at CpG dinucleotides in the promoter region of the genes is a major epigenetic modification in mammalian genomes that can have profound effects on gene expression. The PTPL1 methylation pattern was screened by methylation-specific polymerase chain reaction (MSP) in 7 lymphoma-derived cell lines and in 47 samples of diffuse large B cell lymphoma (DLBCL). The PTPL1 gene was hypermethylated in the CA46, Raji, Jurkat and DB cell lines; however, it was unmethylated in the Hut78, Maver and Z138 cell lines. The expression of PTPL1 mRNA was re-inducible by 5-azacytidine (5-Aza), an agent of DNA demethylation. The methylations were detected in 59.6% of DLBCL versus 6.3% in reactive lymph node proliferation. Therefore, the present data showed that PTPL1 was epigenetically regulated in NHL suggesting an involvement of the PTPL1 tumor-suppressor genes in NHL, and highlights 5-Aza as a potential therapeutic candidate for NHL.

GC-02 * CNS GERMINOMAS ARE CHARACTERIZED BY GLOBAL DEMETHYLATION, CHROMOSOMAL INSTABILITY AND MUTATIONAL ACTIVATION OF THE KIT-, RAS/RAF/ERK- AND AKT-PATHWAYS

CNS germinomas represent a unique germ cell tumor entity characterized by uniform undifferentiated tumor cells and a high response rate to current treatment protocols. However, only limited information is available on their underlying genomic, epigenetic and biological alterations. In this study we perfomed a genome-wide analysis of genomic copy number alteration and allelic disbalances in 37 cases of CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry with a specific antibody against methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members using FFPE derived DNA. Ras/Erk and Akt pathway activation was analysed by immunostaining with specific antibodies against phospho-Erk and phospho-Akt. All germinomas coexpressed Oct4 and Kit but showed an extensive global demethylation of nuclear DNA compared to other germ cell tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in 32/37 tumors with a high frequency of regional gains and losses including high level amplifications. Activating mutations of KIT exons 11, 13, 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS, RRAS2 and RHOH as well as mutation of BRAF indicated mutational activation of crucial signalling pathways. Activation of both, Ras/Erk and Akt pathways was demonstrated by immunohistochemistry of phospho-Erk and phospho-Akt in germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding is associated with extensive genomic instability in the majority of germinomas. In addition, mutational activation of KIT, Ras/Raf/Erk- and Akt- pathways is characteristic indicating the biological importance of these pathways and potential targets for therapy.

Effects of aging and walnut‐rich diet on DNA methylation and expression of immediate‐early genes in critical brain regions.

Emerging evidence indicates a direct link between age-associated changes in epigenetic mechanisms and onset of neurodegenerative diseases, and that these genomic modulations are directly affected by the diet. Diets deficient in folate, choline and methionine, or the trace elements zinc and selenium, are reported to induce DNA hypomethylation and are linked to aberrant gene transcription. In a cognitively healthy adult, transcription of immediate-early genes (IEGs), e.g., Arc(activity-regulated cytoskeletal gene),zif268(nerve growth factor inducible-A),bdnf(brain-derived neurotrophic factor) and Reln (plasticity-promoting Reelin) are essential in memory formation and synaptic plasticity. In the current study, we investigated the effect of walnut diets, which are rich in polyunsaturated fatty acids and nutrients such as folates, selenium, magnesium and polyphenolics, on epigenetic mechanisms in critical regions of the brain. Young (3 months, n=30, 10/group) and old (19 months, n=45, 15/group) male Fischer 344 rats were supplemented with control (0%), 6% or 9% walnut diets for 10 weeks. Using LC-MS, absolute amounts of deoxycytidine and methylated cytosine residues in genomic DNA were quantified. Using RT-PCR, expression of bdnf, zif268, reln and arc were measured in hippocampus and frontal cortex. Significant aging effects (p<0.05) were observed for the expression of bdnf, zif268, reln and arc. The 9% walnut diet significantly (p<0.01) increased the expression of zif268 in the hippocampus compared to control animals. The results suggest that dietary walnut may have protective effects on the aging brain. Supported by USDA Intramural and California Walnut Commission.

The tRNA Methylome of Trypanosoma brucei, the Causative Agent of African Sleeping Sickness

Trypanosoma brucei is the causative agent of African Sleeping Sickness. The regulation of gene expression in T. brucei primarily occurs at the posttranscriptional level, indicating that RNA processing and modification events are extremely important for the parasite's life cycle. Yet, there is little information regarding the collection of covalent RNA modifications that comprise the epitranscriptome in this organism or any protozoan parasite. We focused on cytosine base modifications of tRNAs, as tRNAs are well known to contain a range of modified RNA bases, including 5‐methylcytosine. Small RNAs were isolated from insect‐stage T. brucei parasites and unmodified cytosines were converted to uracils using sodium bisulfite treatment. Sodium bisulfite‐treated small RNAs were analyzed by RNAseq. To confirm the RNAseq data, sodium bisulfite‐treated RNA samples were amplified by RT‐PCR from either total RNA or small RNA fractions, and analyzed by digestion with HpaII or via Sanger sequencing. Overall, our data indicate that tRNA molecules contain between zero to four 5‐methylcytosine residues. The most common location of 5‐methylcytosine in T. brucei tRNA is at the junction between the variable region and TψC arm at positions C48, C49, and C50, although different tRNAs have different numbers of 5‐methylcytosines in this region. There was little evidence for methylation of other sites in the tRNA, including C38 methylation in the anticodon loop. Overall, our data indicated that T. brucei tRNAs contain 5‐methylcytosine at some, but not all standard eukaryotic positions, and the levels of 5‐methylcytosine vary in different tRNA molecules.

Aberrant DNA Methylation and Genome Instability and Mutation in Cancer

In the process of normal cells transforming into cancer cells with the disruption control of epigenetic and genetic, they will gradually acquire the cancer Hallmarks. Epigenetic and genetic influence each other and closely cooperate to promoter oncogenic transformation in variety ways. It is clear that DNA methylation plays important roles in generation of mutation of tumor suppressor genes (TSGs) eventually lead to inactivation such as the probability of C mutation to T of the well-known TSGs p53 is high due to 5-methylcytosine residues is more prone to spontaneous deamination. The most common pattern of epigenetic control of tumor suppressor genes inactivation is hypermethylation of promoter region in cancers and the hypermethylation of CpG islands can also contribute to the increasing mutation rate of tumor suppressor genes such as CpG islands hypermethylation appeared to be tightly linked with the V600E mutation of the BRAF oncogene in colorectal cancer. Global hypomethylation is closely associated with chromosomal instability, which the methylation status of LINE-1 is a marker of global methylation, there is a significant relationship between LINE-1 hypomethylation and DNA copy number variation in the gastrointestinal stromal tumor. Aberrant DNA methylation is pervasive in cancer, which is similar to genomic instability and mutation.

IDH Mutations, 2-Oxoglutarate-dependent Dioxygenases, and Leukemia

Somatic isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations have been discovered in a variety of neoplasms, including glioblastomas and acute myelogenous leukemias. Cancer-associated IDH mutants produce large quantities (low mM) of the R enantiomer of 2-hydroxyglutarate (R-2HG). R-2HG resembles 2-oxoglutarate, which is a Krebs Cycle intermediate and an essential cofactor for a variety of enzymes including the ~70 2-oxoglutarate-dependent dioxygenases. Included among these enzymes are the EglN prolyl hydroxylases that earmark the hypoxia-inducible factor (HIF) for destruction under normoxic conditions, the JmjC histone demethylases, and the ten-eleven translocation (TET) enzymes that hydroxylate methylcytosine residues. In general, both R-2HG and the alternative enantiomer, S-2HG, inhibit 2-oxoglutarate-dependent dioxygenases, with the (S) enantiomer typically being more potent, at least as measured in vitro. We recently discovered that R-2HG, in contrast to S-2HG, potentiates, rather than inhibits, EglN activity; we linked this, and subsequent downregulation of HIF, to the ability of mutant IDH1 to transform human astrocytes. Moreover, we found that R-2HG, but not S-2HG, is sufficient to promote leukemic transformation and that its effects are reversible. In this setting, inactivation of EglN, as occurs with S-HG, appears to be antithetical to transformation. R-2HG promotes leukemic transformation by inhibiting targets such as TET2, while sparing EglN activity. Recent clinical data show that inhibiting R-2HG production can induce complete remissions in some patients with IDH mutant leukemias. We are currently trying to identify the relevant downstream targets of TET2 in IDH mutant leukemias and are also exploring whether loss of EglN activity has antileukemic effects in mouse models in vivo. Finally, we are exploring whether any other 2-oxoglutarate-dependent dioxygenases, such as the RBP2/JARID1A/KDM5A, might be viable drug targets in leukemia. DisclosuresKaelin:Agios: Consultancy, Membership on an entity’s Board of Directors or advisory committees; FibroGen: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Lilly: Membership on an entity’s Board of Directors or advisory committees; Nextech: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Peloton: Consultancy, Membership on an entity’s Board of Directors or advisory committees; TRACON: Consultancy.