DNA Methylation Regulates Gene Expression Research Articles

Abstract 4124828: The Methylation Landscape Of Human Aortic And Mitral Valvular Interstitial Cells

Valvular interstitial cells (VICs), the valves’ predominant cell type, are crucial in ensuring proper valve function, structural integrity, tissue repair and valve homeostasis. DNA methylation regulates gene expression profiles and a matched aortic to mitral human VIC methylation analysis will further our understanding of aortic- and mitral-specific VIC biology and pathobiology. This is the first study analyzing methylation profiles of non-diseased, matched human aortic and mitral VICs. We analyzed the differential methylation fingerprint of 12 non-diseased aortic and mitral VICs (10 males:2 females; 42–64 years) extracted from de-endothelialized leaflets using reduced representation bisulfite sequencing (RRBS) on HiSeq2500. Analysis of methylation levels of 12,676 genome-wide promoters revealed 651 differentially methylated (DM) promoters. The top 3 DM promoters were linked to TBX4 (meth diff = -28.9%, P = 5.2E-135) essential for valve morphogenesis, to EXOC3L2 (meth diff = 25.7%, P = 1.3E-92) and NT5DC2 (meth diff = -20.3%, P = 7.1E-69) potentially implicated in ECM remodeling, the former via vesicle trafficking and the latter through nucleotide metabolism. Functional classification and network analysis showed that the genes associated with the DM promoters were enriched for WNT-, TGFβ-, FGF-, EGF- and PDGF- pathways involved in physiological processes such as valve development, repair and VIC regulation, as well as pathological processes such as calcification. Additional enrichment was detected for integrin- and cadherin- pathways involved in cell-cell-ECM interactions and endothelin- and VEGF pathways involved in hemodynamic regulation. This work provides the first insight into the differential methylation regulation patterns of human aortic and mitral VICs. Understanding how aortic and mitral VICs are differentially epigenetically regulated will extend our understanding of their role in aortic- and mitral-specific leaflet homeostasis, susceptibility to disease processes and will contribute to dynamic innovative experimental setup designs, animal-free human-based modeling frameworks, tissue engineering and drug identification approaches.

The OsAGO2-OsNAC300-OsNAP module regulates leaf senescence in rice.

Leaves play a crucial role in the growth and development of rice (Oryza sativa) as sites for the production of photosynthesis. Early leaf senescence leads to substantial drops in rice yields. Whether and how DNA methylation regulates gene expression and affects leaf senescence remains elusive. Here, we demonstrate that mutations in rice ARGONAUTE 2 (OsAGO2) lead to premature leaf senescence, with chloroplasts in Osago2 having lower chlorophyll content and an abnormal thylakoid structure compared with those from wild-type plants. We show that OsAGO2 associates with a 24-nt microRNA and binds to the promoter region of OsNAC300, which causes DNA methylation and suppressed expression of OsNAC300. Overexpressing OsNAC300 causes the similar premature leaf senescence as Osago2 mutants and knocking out OsNAC300 in the Osago2 mutant background suppresses the early senescence of Osago2 mutants. Based on yeast one-hybrid, dual-luciferase, and electrophoresis mobility shift assays, we propose that OsNAC300 directly regulates transcription of the key rice aging gene NAC-like, activated by APETALA3/PISTILLATA (OsNAP) to control leaf senescence. Our results unravel a previously unknown epigenetic regulatory mechanism underlying leaf senescence in which OsAGO2-OsNAC300-OsNAP acts as a key regulatory module of leaf senescence to maintain leaf function.

O-028 DNA methylation testing with cervical secretion can serve as a non-invasive biomarker for predicting successful implantation in frozen-thawed embryo transfer

Abstract Study question Is cervical secretion gene methylation profile associated with embryo implantation outcome, and can be used as a non-invasive biomarker for pregnancy prediction in FET cycles? Summary answer Cervical secretion gene methylation profiles show significant associations with implantation outcomes, and can serve as a non-invasive predictor of successful pregnancy in FET cycles. What is known already Cause of implantation failure remains a black box in reproductive medicine, and the exact mechanism of how endometrial receptivity is still behind the curtain. Epigenetic modifications including DNA methylation regulate gene expression and may alter endometrium receptivity. Endometrial DNA methylation changes vigorously only during the reconstruction of a new endometrium throughout each menstrual cycle, suggesting a unique methylation landscape in each regenerated endometrium. Cervical secretions contain implantation-related cytokines and genetic material from endometrium, and the DNA methylation profiles of cervical secretions is highly correlated with endometrium. Therefore, cervical secretions can be used as non-invasive surrogates for investigating endometrial condition. Study design, size, duration This multi-centered, case-control study enrolled 72 infertile females who entered the FET cycle with at least one high-quality blastocyst available between Aug 2017 and Dec 2022. Embryos assessed by Gardner scoring system with grade 3BB or above were defined as high-quality. Cervical secretions were collected on the day right before embryo transfer, and then DNA methylation profile were assessed. Participants/materials, setting, methods These females aged between 20-45 years-old with regular menstruation cycle (24-35 days) and normal BMI (17.5–32.0 kg/m2). Uterine cavity lesions and hydrosalpinx without treatment, adenomyosis, large uterine myoma that distorted endometrium, and thin endometrium (<7mm) were excluded. The association between the cervical secretion gene methylation profile and pregnancy outcome was evaluated via quantitative methylation-specific PCR (qMSP), methylation array, and genome-wide analysis. Different machine learning algorithms were utilized for differential methylation analysis and implantation prediction. Main results and the role of chance Cervical secretion DNA methylome differed between receptive (pregnant) and non-receptive (non-pregnant) FET cycle with genome-wide methylation array. The accuracy of pregnancy outcome prediction using top 2000 differential methylation probes (DMPs) in promoter region can be as high as 86% in all tested sample and 96.4% in pregnant samples. Bioinformatic analysis of six selected candidate genes related to implantation from literature review showed significant differences in DNA methylation between receptive and non-receptive endometrium. The methylation level of the six candidate genes with eight regions were measured by the qMSP platform and showed varying degrees of differential methylation status between the pregnancy and non-pregnancy groups. The AUCs for a single gene/region ranged from 0.53–0.67 for implantation prediction. Ten different machine learning models were applied and the results showed that the combination of candidate genes’ DNA methylation profiles could differentiate pregnant from non-pregnant samples with an accuracy as high as 86.67% and an AUC of 0.81 by logistic regression model. The best model from different machine learning algorithms using selected DMPs of genes related to window of implantation (WOI) can predict ongoing pregnancy with accuracy rates as high as 86.78% and AUC values up to 0.91. Limitations, reasons for caution The results obtained from a relatively small cohort size, and the timing of sample collection is on the day right before embryo transfer. Further investigation of methylation profiles in late follicular phase along with large sample size with euploid embryo transfer are mandatory for validation and clinical applications. Wider implications of the findings We firstly demonstrated the feasibility of cervical secretion methylation profile as a non-invasive testing for pregnancy prediction. The promising result may contribute to predicting endometrial receptivity for cycles with a favorable endometrium and maximize the successful pregnancy rate of embryo transfer. Trial registration number not applicable

Methylation levels of the IL10 gene in peripheral blood are related to the intractability of Graves' disease

The prognosis of autoimmune thyroid diseases (AITDs), including Hashimoto's disease (HD) and Graves' disease (GD), is difficult to predict. DNA methylation regulates gene expression of immune mediating factors. Interleukin (IL)-10 is a Th2 cytokine that downregulates inflammatory cytokines produced by Th1 cells. To clarify the role of methylation of the IL10 gene in the prognosis of AITD, we evaluated the methylation levels of two CpG sites in the IL10 promoter using pyrosequencing. The methylation levels of the −185 CpG site of the IL10 gene were related to age and GD intractability in GD patients. Furthermore, the C carrier of the IL10–592 A/C polymorphism was related to low methylation levels of the −185 CpG site. The methylation levels of the IL10–185 CpG site of the IL10 gene were related to the intractability of GD and were lower in individuals with the C allele of the IL10–592 A/C polymorphism.

Wemics: A Single-Base Resolution Methylation Quantification Method for Enhanced Prediction of Epigenetic Regulation.

DNA methylation, an epigenetic mechanism that alters gene expression without changing DNA sequence, is essential for organism development and key biological processes like genomic imprinting and X-chromosome inactivation. Despite tremendous efforts in DNA methylation research, accurate quantification of cytosine methylation remains a challenge. Here, a single-base methylation quantification approach is introduced by weighting methylation of consecutive CpG sites (Wemics) in genomic regions. Wemics quantification of DNA methylation better predicts its regulatory impact on gene transcription and identifies differentially methylated regions (DMRs) with more biological relevance. Most Wemics-quantified DMRs in lung cancer are epigenetically conserved and recurrently occurred in other primary cancers from The Cancer Genome Atlas (TCGA), and their aberrant alterations can serve as promising pan-cancer diagnostic markers. It is further revealed that these detected DMRs are enriched in transcription factor (TF) binding motifs, and methylation of these TF binding motifs and TF expression synergistically regulate target gene expression. Using Wemics on epigenomic-transcriptomic data from the large lung cancer cohort, a dozen novel genes with oncogenic potential are discovered that are upregulated by hypomethylation but overlooked by other quantification methods. These findings increase the understanding of the epigenetic mechanism by which DNA methylation regulates gene expression.

Genome-Wide DNA Methylation Analysis and Functional Validation of Litter Size Traits in Jining Grey Goats.

DNA methylation (DNAm) is associated with the reproductive system. However, the genetic mechanism through which DNAm regulates gene expression and thus affects litter size in goats is unclear. Therefore, in the present work, genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues were comprehensively analyzed via WGBS, and RNA-Seq data were combined to identify candidate genes associated with litter size traits in the Jining Grey goat. Finally, BSP and RT-qPCR were used to verify the sequencing results of the key genes. Notably, the DNMT genes were downregulated at the expression level in the HP group. Both groups exhibited comparable levels of methylation. A total of 976 differentially methylated regions (DMRs) (973 DMRs for CG and 3 DMRs for CHG) and 310 differentially methylated genes (DMGs) were identified in this study. Through integration of WGBS and RNA-Seq data, we identified 59 differentially methylated and differentially expressed genes (DEGs) and ultimately screened 8 key DMGs (9 DMRS) associated with litter size traits in Jining Grey goats (SERPINB2: chr24_62258801_62259000, NDRG4: chr18_27599201_27599400, CFAP43: chr26_27046601_27046800, LRP1B. chr2_79720201_79720400, EPHA6: chr1_40088601_40088800, TTC29: chr17_59385801_59386000, PDE11A: chr2_117418601_117418800 and PGF: chr10_ 16913801_16914000 and chr10_16916401_16916600). In summary, our research comprehensively analyzed the genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues. The data findings suggest that DNAm in goat ovaries may play an important role in determining litter size.

Joint Analysis of Genome-wide DNA Methylation and Transcription Sequencing Identifies the Role of BAX Gene in Heat Stress-Induced-Sertoli Cells Apoptosis.

The problem of male infertility is a global health crisis and poses a serious threat to the well-being of families. Under heat stress (HS), the reduction of Sertoli cells (SCs) inhibits energy transport and nutrient supply to germ cells, leading to spermatogenesis failure. DNA methylation of genes is a central epigenetic regulatory mechanism in mammalian reproduction. However, it remains unclear how DNA methylation regulates gene expression in heat-stressed SCs. In this study, we investigated whether the decrease in SC levels during HS could be related to epigenetic DNA modifications. The cells exposed to HS showed changes in differential methylation cytosines and regions (DMCs/DMRs) and differential expression genes (DEGs), but not in global DNA methylations. One of the most important biological processes affected by HS is cell apoptosis induced by the intrinsic apoptotic signaling pathway (GO: 2,001,244, P < 0.05) by enrichment in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The joint analysis showed that several gene expressions in RNA-seq and WGBS overlapped and the shortlisted genes BAX, HSPH1, HSF1B, and BAG were strongly correlated with stress response and apoptosis. Methylation-specific PCR (MSP) and flow cytometry (FCM) analyzes showed that reduced promoter methylation and enhanced gene expression of BAX with a consequence of apoptosis. The activity of BAX, as well as an increase in its expression, is likely to result in a reduction of SCs population which could further impair ATP supply and adversely affect membrane integrity. These findings provide novel insights into the molecular mechanisms through which stressors cause male reproductive dysfunction and a new molecular etiology of male infertility.

Host DNA Demethylation Induced by DNMT1 Inhibition Up-Regulates Antiviral OASL Protein during Influenza a Virus Infection.

Influenza A virus (IAV) is a leading cause of human respiratory infections and poses a major public health concern. IAV replication can affect the expression of DNA methyltransferases (DNMTs), and the subsequent changes in DNA methylation regulate gene expression and may lead to abnormal gene transcription and translation, yet the underlying mechanisms of virus-induced epigenetic changes from DNA methylation and its role in virus-host interactions remain elusive. Here in this paper, we showed that DNMT1 expression could be suppressed following the inhibition of miR-142-5p or the PI3K/AKT signaling pathway during IAV infection, resulting in demethylation of the promotor region of the 2'-5'-oligoadenylate synthetase-like (OASL) protein and promotion of its expression in A549 cells. OASL expression enhanced RIG-I-mediated interferon induction and then suppressed replication of IAV. Our study elucidated an innate immunity mechanism by which up-regulation of OASL contributes to host antiviral responses via epigenetic modifications in IAV infection, which could provide important insights into the understanding of viral pathogenesis and host antiviral defense.

B cell Tet2 promotes maturation of the antibody response through active DNA demethylation and OGT-mediated histone glycosylation of Aicdaand Prdm1loci

Abstract Epigenetic DNA methylation regulates gene expression and, therefore, cellular functions. It typically represses gene transcription, which can be re-activated by demethylation in the absence of cell division by the recently characterized Tet1, Tet2 and Tet3 dioxygenases. Tet2, the most abundant Tet protein in B cells, not only actively demethylates DNA but also promotes Ogt recruitment to chromatin to effect Ogt-dependent histone O-GlcNAcylation. This potentiates catalytic DNA demethylation to activate gene expression. To define the role of Tet2 in B cell functions and antibody responses, we generated Aicda creTet2 fl/flmice, which specifically delete Tet2 in activated B cells. Aicda creTet2 fl/flmice showed significantly reduced antibody responses to T-dependent and T-independent antigens, due to downregulation of AID and Blimp-1 expression, CSR/SHM and plasma cell differentiation. Accordingly, activated Aicda creTet2 fl/flB cells reduced DNA hydroxymethylation (resulting in increased methylation) and histone O-GlcNAcylation of Aicda and Prdm1 promoters and enhancers, thereby dampening CSR/SHM and plasma cell differentiation. These were partially rescued by enforced expression of Tet2 mutants defective in catalytic activity or Ogt-binding, but not by a double mutant lacking both activities. Accordingly, Tet2 and Ogt inhibitors synergistically inhibited Aicda and Prdm1 expression in both human and mouse B cells. Conversely, Tet2 activation by vitamin C enhanced CSR and plasma cell differentiation. Thus, Tet2 plays a B cell-intrinsic role in modulation of CSR/SHM and plasma cell differentiation through catalytic active DNA demethylation and (non-catalytic) Ogt-mediated histone O-GlcNAcylation. Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to PC.

The prognostic value and immune correlation of IL18 expression and promoter methylation in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is not sensitive to immunotherapy and has poor prognosis. DNA methylation regulates gene expression, and its abnormal changes are related to many human diseases. Recently, DNA methylation has been found to participate in immune infiltration in various cancers. However, its pattern in RCC remains poorly understood.ResultsWe found that IL18 was significantly over-expressed in RCC tumor tissues compared to normal adjacent tissues The IL18 promoter region was hypomethylated, which was strongly correlated with elevated IL18 mRNA expression, and predicted advanced clinicopathological characteristics and shorter overall survival. Furthermore, we found that IL18 promoter methylation was significantly related to the down-regulation of immune checkpoint molecules and increase of CD8 + T cell infiltration in RCC tumor tissues.ConclusionsWe have identified the important role of IL18 promoter methylation and expression, which are associated with clinicopathological characteristics, overall survival, immune cell infiltration and expression of immune checkpoint molecules in RCC. We present the rationale for IL18 promoter methylation as a molecular biomarker for predicting the response of RCC to immune checkpoint inhibitors.

Geminiviruses boost active DNA demethylation for counter-defense.

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

Epigenetic and Genetic Contribution for Expression Bias of Homologous Alleles in Polyploid Sugarcane

DNA methylation regulates gene expression in eukaryotes, but their roles in gene expression changes in polyploids are poorly understood. Here, we comparatively analyzed the DNA methylation profiles and transcriptome maps of four tissues (leaf, rind, pith, and root) in autopolyploid sugarcane (Saccharum spontaneum). The overall DNA methylation levels were relatively equal and were consistent with the similar genome-wide expression levels of homologous alleles among different homologous chromosomes. However, tetrad alleles showed a large proportion of non-balanced alleles (85–89%) compared with balanced alleles (11–15%). For tetrad alleles, highly-expressed alleles showed lower Ka/Ks values than those in lowly-expressed alleles, indicating that dominant (and non-suppressed) alleles were under strong purifying selection pressure compared to non-dominant (and suppressed) alleles. Specifically, higher DNA methylation levels were found in non-balanced alleles compared to balanced alleles, as well as a higher CG to TG substitution rate, suggesting epigenetic and genetic variation associated with transcription divergence. Moreover, among non-balanced alleles, which were associated with DNA methylation changes, many were involved in several processes relevant to agronomic traits, such as responses to stress and carbohydrate transport. Taken together, our results provide unique epigenetic insights into the transcriptional regulation of homologous alleles, which contribute to the key agronomic traits in sugarcane and facilitate epigenetic studies in other polyploid crops.

Characterization of the Clinical and Molecular Perspectives of Epigenetics

This review provides the latitude to examine the extant information in the univeral characterizations of epigenetic formulations. Epigenetics encompasses the interaction of behaviours and environment culminating in changes which influence gene activity. In contrast to genetic modifications, epigenetic modifications are reversible; and do not alter the DNA sequence but are capable of interferring in the way the DNA sequence is read. Epigenetic alterations involve genetic changes which effect gene functionality without modifying the underlying DNA sequence. DNA methylation depicts the covalent superimposed methyl group to cytosine in CpG dinucleotides. DNA methylation presents as a veritable epigenetic modification; and it governs gene expression by changing chromosome structure, DNA conformation and stability as well as the function trajectory between DNA and protein. DNA methylation regulates gene expression via the conscription of proteins associated with gene expression or by the inhibition of the binding of transcription factor(s) to DNA. Whereas genetic alterations do modify protein formation, it is clear that epigenetic alterations impact on gene expression to put genes ''on'' and ''off'', as appropriate. The resultant impact of environmental and anthropogenic idiosyncracies, such as diet and physical activity are liable to induce epigenetic modifications in behaviours and gene-environment interactions. Genes are not always in functional mode. DNA methylation constitutes a select epigenetic process applied by cells for the control of gene expression. within the genome. Alterations in gene activity and epigenetic errors can result in varying genetic, metabolic and degenerative disorders which may disparately or in comorbid presentaions influence inter alia health, gene activity or expression, protein production and functionality. This entry exemplifies the reading and understanding of epigenetics in relation to inter alia beneficial developmental theories within the human race.

Influence of DAT1 Promotor Methylation on Sports Performance.

In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factors (TFs) to DNA. As there are still many questions concerning the role of methylation in creating personality, we concentrated on searching for such associations. The research group was 100 sports male subjects (mean age = 22.88, SD = 6.35), whereas the control group included 239 healthy male volunteers matched for age (mean age = 21.69, SD = 3.39), both of European origin. The methods used in our research were as follows: DNA isolation, methylation-specific PCR, sequencing chromatophores, all conducted according to the manufacturer’s procedure. To evaluate personality traits, the NEO Five-Factor Personality Inventory (NEO-FFI) and STAI Inventory were used. We observed the existence of a statistically significant correlation for all the aspects of personality covered and CpG islands’ methylation. Nonetheless, we think that the tested group and the number of tested promotor islands in the DAT1 gene are still too small to make explicit conclusions, so it needs further profound analysis.

MRNA Expressions of Methylation Related Enzymes and Duration of Thermal Conditioning in Chicks.

DNA methylation regulates gene expression by modifying the nucleosome structure of DNA, without altering the gene sequence. It has been reported that DNA methylation reactions are catalyzed by several enzymes. In chickens, thermal conditioning treatment affects the central DNA methylation levels. The purpose of this study was to clarify the changes in DNA methylation and demethylation factors during thermal conditioning in the hypothalamus of 3-day-old chicks. Male chicks (3-days old) were exposed to 40±0.5°C as a thermal conditioning treatment for 1, 2, 6, 9, or 12 h. The control chicks were kept in a thermoneutral zone (30±0.2°C). After thermal conditioning, the mRNA levels of DNA methyltransferase (DNMT)-1, -3a, -3b, and ten-eleven translocation (TET)-1, -2, and -3 in the hypothalamus were measured by q-PCR. The mRNA levels of DNMT-3a and TET-1 were increased by thermal conditioning. Moreover, the expression level of TET-1 increased with the loading time of the thermal conditioning. The gene expressions of DNMT-1, DNMT-3b, TET-2, and TET-3 were not affected by thermal conditioning. Since DNMT-3a is a catalyst for de-novo DNA methylation and TET-1 catalyzes the oxidation of methylated cytosine, it is suggested that the thermal conditioning increased the activation of DNA methylation and demethylation factors, which occur in the hypothalamus of neonatal chicks.

Computational methods and next-generation sequencing approaches to analyze epigenetics data: Profiling of methods and applications.

Epigenetics is mainly comprised of features that regulate genomic interactions thereby playing a crucial role in a vast array of biological processes. Epigenetic mechanisms such as DNA methylation and histone modifications influence gene expression by modulating the packaging of DNA in the nucleus. A plethora of studies have emphasized the importance of analyzing epigenetics data through genome-wide studies and high-throughput approaches, thereby providing key insights towards epigenetics-based diseases such as cancer. Recent advancements have been made towards translating epigenetics research into a high throughput approach such as genome-scale profiling. Amongst all, bioinformatics plays a pivotal role in achieving epigenetics-related computational studies. Despite significant advancements towards epigenomic profiling, it is challenging to understand how various epigenetic modifications such as chromatin modifications and DNA methylation regulate gene expression. Next-generation sequencing (NGS) provides accurate and parallel sequencing thereby allowing researchers to comprehend epigenomic profiling. In this review, we summarize different computational methods such as machine learning and other bioinformatics tools, publicly available databases and resources to identify key modifications associated with epigenetic machinery. Additionally, the review also focuses on understanding recent methodologies related to epigenome profiling using NGS methods ranging from library preparation, different sequencing platforms and analytical techniques to evaluate various epigenetic modifications such as DNA methylation and histone modifications. We also provide detailed information on bioinformatics tools and computational strategies responsible for analyzing large scale data in epigenetics.

Catalytically inactive Dnmt3b rescues mouse embryonic development by accessory and repressive functions

DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.

Determination of the presence of 5-methylcytosine in Paramecium tetraurelia.

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.

Epigenetic DNA Methylation Mediating Octopus vulgaris Early Development: Effect of Essential Fatty Acids Enriched Diet.

The common octopus, Octopus vulgaris, is a good candidate for aquaculture but a sustainable production is still unviable due to an almost total mortality during the paralarvae stage. DNA methylation regulates gene expression in the eukaryotic genome, and has been shown to exhibit plasticity throughout O. vulgaris life cycle, changing profiles from paralarvae to adult stages. This pattern of methylation could be sensitive to small alterations in nutritional and environmental conditions during the species early development, thus impacting on its health, growth and survival. In this sense, a full understanding of the epigenetic mechanisms operating during O. vulgaris development would contribute to optimizing the culture conditions for this species. Paralarvae of O. vulgaris were cultured over 28 days post-hatching (dph) using two different Artemia sp. based diets: control and a long chain polyunsaturated fatty acids (LC-PUFA) enriched diet. The effect of the diets on the paralarvae DNA global methylation was analyzed by Methyl-Sensitive Amplification Polymorphism (MSAP) and global 5-methylcytosine enzyme-linked immunosorbent assay (ELISA) approaches. The analysis of different methylation states over the time revealed a global demethylation phenomena occurring along O. vulgaris early development being directly driven by the age of the paralarvae. A gradual decline in methylated loci (hemimethylated, internal cytosine methylated, and hypermethylated) parallel to a progressive gain in non-methylated (NMT) loci toward the later sampling points was verified regardless of the diet provided and demonstrate a pre-established and well-defined demethylation program during its early development, involving a 20% of the MSAP loci. In addition, a differential behavior between diets was also observed at 20 dph, with a LC-PUFA supplementation effect over the methylation profiles. The present results show significant differences on the paralarvae methylation profiles during its development and a diet effect on these changes. It is characterized by a process of demethylation of the genome at the paralarvae stage and the influence of diet to favor this methylation loss.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line

PurposeDNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. Methods1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. ResultsOur results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). Treatmentwith azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. Conclusions1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest.