Altered DNA Methylation Status Research Articles

P02.03.A INTEGRATIVE (EPI)GENETIC ANALYSIS OF INVASION-PROMOTING GENES IN HIGH-GRADE GLIOMA CELLS

Abstract BACKGROUND DNA methylation-based analyses of brain tumors including glioma is currently the gold standard for diagnostic classification. Aberrant epigenetic alterations can lead to inappropriate gene expression and thereby promote tumorigenesis. Glioblastoma (GBM), CNS WHO grade 4, is characterized by a very diffuse infiltrative growth into the adjacent brain parenchyma. Due to these highly invasive but also genetically heterogeneous and plastic cellular phenotypes, standard treatment, including maximum resection, radio- and chemotherapy, has limited efficacy. It can be assumed that intertumoral or molecular heterogeneity of gliomas on transcriptional level is caused by altered epigenetic changes within the individual tumor cells. Considering this, we hypothesize that tumor cell invasion being one of the critical hallmarks of highly malignant gliomas goes along with altered DNA methylation status and following from this changes in invasion-promoting transcript expression. MATERIAL AND METHODS We performed several in vitro screening approaches to describe the genetic, epigenetic and transcriptional status of invasion glioma cells. Within our first approach, we isolated fractions of glioma cells with increased invasive properties and performed Illumina® array-based methylation profiling as well as RNA sequencing analysis to identify methylation-driven, invasion-associated gene sets. During a second approach, the inducible CRISPR/Cas9 Synergistic Activation Mediator (SAM) system and a genome-wide sgRNA library were introduced in a serum-free glioma cell line. During CRISPR/Cas9 SAM mediated single-gene activation cells were subjected to a transwell invasion assay and sgRNA representation within the invasive glioma cell fraction was determined by next-generation-sequencing (NGS). RESULTS In this study, we integrated Illumina HumanMethylation850K methylation data, RNA-sequencing data and invasion-promoting sgRNA representation data. By systemic screening we identified an invasion-promoting gene signature and the correlating promotor DNA-methylation status. Future studies will be directed to further investigate the impact of identified genes in invasion of high-grade glioma cells. CONCLUSION This integrative experimental approach complements existing genome-wide inhibitory screens as well as incorporates for the first time DNA-methylation profiles and transcriptome analysis with gene-sets identified by CRISPR/Cas9 activation screen and thereby holds the potential to identify novel invasion-essential candidate genes.

Exposure to war and conflict: The individual and inherited epigenetic effects on health, with a focus on post-traumatic stress disorder.

War and conflict are global phenomena, identified as stress-inducing triggers for epigenetic modifications. In this state-of-the-science narrative review based on systematic principles, we summarise existing data to explore the outcomes of these exposures especially in veterans and show that they may result in an increased likelihood of developing gastrointestinal, auditory, metabolic and circadian issues, as well as post-traumatic stress disorder (PTSD). We also note that, despite a potential "healthy soldier effect", both veterans and civilians with PTSD exhibit the altered DNA methylation status in hypothalamic-pituitary-adrenal (HPA) axis regulatory genes such as NR3C1. Genes associated with sleep (PAX8; LHX1) are seen to be differentially methylated in veterans. A limited number of studies also revealed hereditary effects of war exposure across groups: decreased cortisol levels and a heightened (sex-linked) mortality risk in offspring. Future large-scale studies further identifying the heritable risks of war, as well as any potential differences between military and civilian populations, would be valuable to inform future healthcare directives.

Immunity of turbot Induced by inactivated vaccine of Aeromonas salmonicida from the perspective of DNA methylation.

DNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear. In this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida. A total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene. We jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Paternal exposure to arsenic and sperm DNA methylation of imprinting gene Meg3 in reproductive-aged men.

Prenatal exposure to arsenic and mercury have been associated with adverse pregnancy outcomes that might be in part mediated by dynamic modification of imprinting gene that are emerging mechanism. The objective of this study was to examine the impacts of paternal exposure to arsenic and co-exposure to arsenic and mercury on human sperm DNA methylation status of imprinting genes, respectively. A total of 352 male subjects (23-52years old) were recruited and demographic data were obtained through questionnaires. Urinary arsenic and mercury levels were measured using hydride generation-atomic fluorescence spectrometer. Multivariate regression model was employed to investigate the relationship between urinary arsenic levels and sperm DNA methylation status at H19, Meg3 and Peg3, measured by pyrosequencing, and evaluating the interaction with mercury. After adjusting potential confounds factors by multivariate regression model, the results indicated a significantly positive relationship between urinary arsenic levels and the methylation status of Meg3 at both mean level (β = + 0.125, p < 0.001) and all individual CpGs, i.e., CpG1 (β = + 0.094, p < 0.001), CpG2 (β = + 0.132, p < 0.001), CpG3 (β = + 0.121, p < 0.001), CpG4 (β = + 0.142, p < 0.001), CpG5 (β = + 0.111, p < 0.001), CpG6 (β = + 0.120, p < 0.001), CpG7 (β = + 0.143, p < 0.001), CpG8 (β = + 0.139, p < 0.001) of Meg3 DMRs. The interaction effects analysis indicated the interaction effects of arsenic and mercury on Meg3 were not existing. Paternal nonoccupational exposure to arsenic induces the altered DNA methylation status of Meg3 in human sperm DNA. In addition, the interaction effects of arsenic and mercury on Meg3 were not existing. These findings would implicate the sensibility of sperm epigenome for environmental pollutions.

Early Expression of Tet1 and Tet2 in Mouse Zygotes Altered DNA Methylation Status and Affected Embryonic Development.

Ten-eleven translocation (Tet) dioxygenases can induce DNA demethylation by catalyzing 5-methylcytosine(5mC) to 5-hydroxymethylcytosine(5hmC), and play important roles during mammalian development. In mouse, Tet1 and Tet2 are not expressed in pronucleus-staged embryos and are not involved in the genomic demethylation of early zygotes. Here, we investigated the influence of Tet1 and Tet2 on methylation of parental genomes by ectopically expressing Tet1 and Tet2 in zygotes. Immunofluorescence staining showed a marked 5hmC increase in the maternal pronucleus after injection of Tet1 or Tet2 mRNA into zygotes. Whole-genome bisulfite sequencing further revealed that Tet2 greatly enhanced the global demethylation of both parental genomes, while Tet1 only promoted the paternal demethylation. Tet1 and Tet2 overexpression altered the DNA methylation across genomes, including various genic elements and germline-specific differently methylated regions. Tet2 exhibited overall stronger demethylation activity than Tet1. Either Tet1 or Tet2 overexpression impaired preimplantation embryonic development. These results demonstrated that early expression of Tet1 and Tet2 could substantially alter the zygotic methylation landscape and damage embryonic development. These findings provide new insights into understanding the function of Tet dioxygenases and the mechanism of DNA methylation in relation to embryogenesis.

Hyperglycemia Altered DNA Methylation Status and Impaired Pancreatic Differentiation from Embryonic Stem Cells.

The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1, NKX6−1 and NKX6−2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6−2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6−1. Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.

The Tomato ddm1b Mutant Shows Decreased Sensitivity to Heat Stress Accompanied by Transcriptional Alterations.

Heat stress is a major environmental factor limiting crop productivity, thus presenting a food security challenge. Various approaches are taken in an effort to develop crop species with enhanced tolerance to heat stress conditions. Since epigenetic mechanisms were shown to play a regulatory role in mediating plants’ responses to their environment, we investigated the role of DNA methylation in response to heat stress in tomato (Solanum lycopersicum), an important vegetable crop. To meet this aim, we tested a DNA methylation-deficient tomato mutant, Slddm1b. In this short communication paper, we report phenotypic and transcriptomic preliminary findings, implying that the tomato ddm1b mutant is significantly less sensitive to heat stress compared with the background tomato line, M82. Under conditions of heat stress, this mutant line presented higher fruit set and seed set rates, as well as a higher survival rate at the seedling stage. On the transcriptional level, we observed differences in the expression of heat stress-related genes, suggesting an altered response of the ddm1b mutant to this stress. Following these preliminary results, further research would shed light on the specific genes that may contribute to the observed thermotolerance of ddm1b and their possibly altered DNA methylation status.

The Effect of 5-Azacitidine Treatment on the Biologic Characteristics of Bone Marrow Mesenchymal Stem Cells in Patients with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell characterized by ineffective bone marrow (BM) hematopoiesis and increased risk for leukemic evolution. An abnormal BM microenvironment has been reported to contribute to BM failure in MDS through defective support of hematopoiesis. The cellular elements of the BM microenvironment derive from a common progenitor cell, namely the mesenchymal stem cell (MSC). We have previously shown that MSCs from MDS patients display impaired proliferative and clonogenic potential and develop chromosomal alterations during passages.It has been shown that DNA methylation occurs at a high frequency in MDS and that gene reactivation by hypo-methylating agents may have a positive impact on patients' outcome. Recent evidence also suggests altered global DNA methylation status in BM-MSCs of MDS patients. The methyl transferase inhibitor 5-Azacitidine (ΑΖΑ) is widely used for the treatment of MDS; however the effect of AZA on patients' BM microenvironment and specifically on the MSC compartment has not been extensively studied. In the present study we investigate the functional, molecular and cytogenetic characteristics of ex vivo expanded BM-MSCs from MDS patients receiving AZA, before and after 6-cycle therapy.MSCs were isolated and expanded from 14 patients with high risk MDS and 10 age- and sex-matched hematologically healthy subjects undergoing surgery for hip replacement, after informed consent. MSCs were expanded and re-seeded for a total of 5 passages (P) and phenotypically characterized by flow cytometry. MSCs were induced to differentiate to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. MSC growth characteristics were assessed by the population doubling time (PDT) and the methyl-triazolyl-tetrazolium (MTT) assay throughout passages. The clonogenic potential of MSCs was evaluated by the colony forming unit fibroblast assay. Conventional cytogenetic and array comparative genomic hybridization (aCGH) analyses were performed in cultured expanded MSCs at P2.MSCs from MDS patients before and after therapy, displayed normal spindle-shape morphology and immunophenotypic characteristics as shown by the expression of CD90, CD105, CD44, CD29, CD73, and the lack of expression of CD45, CD14 and CD34. MSCs were also able to differentiate into adipocytes and osteoblasts before and after therapy, as evidenced by the Oil Red O and Alizarin Red staining, respectively, and the expression of differentiation specific genes. Cumulative analysis showed that the proliferation potential of patient MSCs, evaluated by the PDT and MTT assays, was statistically significant decreased compared to healthy individuals (P<0.01) but increased significantly following AZA treatment compared to baseline (P<0.05), although not at the level of the healthy controls. A paired analysis of patient samples before and after therapy, also showed that AZA treatment improves significantly the proliferation rate of MDS-MSCs (P<0.05). Similarly, a cumulative and paired analysis showed that the defective (compared to healthy individuals) clonogenic potential of patient MSCs at P2, increased significantly following AZA treatment (P<0.05 and P<0.05, respectively), although not at the level of the healthy individuals. Karyotypic analysis did not reveal any chromosomal abnormalities in MSC samples, before or after AZA treatment. An aCGH analysis of P2 MSCs has - thus far - been performed in three MDS patients before and after AZA therapy. Results have indicated genetic abnormalities (duplications/deletions) in all three samples before treatment that remained unchanged after AZA therapy whereas some new abnormalities also appeared.The data of this ongoing study on the biological properties of MSCs from MDS patients receiving AZA, show an improvement of the clonogenic and proliferative potential of MSCs following treatment. According to aCGH analysis, the genetic abnormalities of MSCs remain unaffected. The findings of the functional and cytogenetic characteristics of patient MSCs before and after therapy will be individually correlated with the MSC methylation status and with patients' outcome and the results are anticipated to provide novel insights in the role of AZA therapy in the BM microenvironment of MDS patients. DisclosuresNo relevant conflicts of interest to declare.

Absence of CG methylation alters the long noncoding transcriptome landscape in multiple species.

The noncoding regions throughout the genome are in large part comprised of transposable elements (TEs), some of which are functionalized with long intergenic noncoding RNAs (lincRNAs). DNA methylation is predominantly associated with TEs, but little is known about its contribution to the transcription of lincRNAs. Here, we examine the lincRNA profiles of DNA methylation-related mutants of five species, Arabidopsis, rice, tomato, maize, and mouse, to elucidate patterns in lincRNA regulation under altered DNA methylation status. Significant activation of lincRNAs was observed in the absence of CG DNA methylation rather than non-CG. Our study establishes a working model of the contribution of DNA methylation to regulation of the dynamic activity of lincRNA transcription.

An altered global DNA methylation status in women with depression

Sparse studies have shown that specific biomarkers of a global DNA methylation status may be related to various mental diseases and states, including: bipolar disorder (BD), anxiety and major depression disorder (MDD). The objective of this study was to analyze potential variation of the above mentioned global methylation status in women with depression. 38 women with a current and clinically confirmed depressive episode suffering from BD type I, type II or MDD and 71 women from the general population and at similar age were recruited for the study. Alu and LINE-1 methylation was assayed with the quantitative methylation-specific PCR technique with TaqMan probes, while the 5-mC and 5-hmC level was determined using the ELISA-based method. Significantly higher levels of 5-mC, Alu and LINE-1 methylation were observed in the women with depression as compared to the controls; while the 5-hmC level revealed to be significantly lower. The BD type I patients presented the highest level of 5-mC of all the women with a depressive episode. 5-mC level in the patients was positively and significantly correlated with the severity of the symptoms of depression. Relationships between Alu or LINE-1 methylation and 5-mC level were statistically significant only in the case of the control women. Alu and LINE-1 methylation do not constitute suitable biomarkers of global DNA methylation in the investigated patients. These findings require confirmation in case-control and prospective epidemiological studies.

Alteration of methylation status in archival DNA samples: A qualitative assessment by methylation specific polymerase chain reaction.

DNA methylation is an epigenetic mechanism that regulates gene expression, which also facilitates genomic imprinting. Genomic imprinting is responsible for differential expression of genes based on parent of origin. Altered methylation of parental alleles results in imprinting disorders diagnosed by methylation specific polymerase chain reaction (MS-PCR) technique. With increasing evidence of genes under epigenetic influence, methylation studies are extensively performed on archival samples. To evaluate effect of storage and storage conditions on DNA methylation, a systematic MS-PCR based analysis was planned on an imprinted gene, SNRPN, located on chromosome 15q11.2. It was assessed by MS-PCR on fresh, 4 -20, and -80°C stored DNA samples for different time periods for systematic evaluation of methylation status. Technical factors like type of sample processing, method of DNA isolation, primer region polymorphism, sample heterogeneity were also evaluated. DNA methylation was observed to be altered for SNRPN gene after storage at -80°C from 2 months onwards. Long-term storage of DNA at -80°C results in altered DNA methylation status. This may lead to false MS-PCR diagnosis of imprinting disorders. Our proof of concept study should be followed with quantitative validation since the findings have critical implications in the present era of biobanking.

MON-LB012 Long-Term Developmental Impact of Withholding Parenteral Nutrition in Pediatric-ICU: A 4-Year Follow-Up of the Pepanic Randomized Controlled Trial

Aim: Between 2012-2015, the PEPaNIC randomized controlled trial, which included 1440 critically ill infants and children, showed that withholding parenteral nutrition during the first week in the pediatric intensive care unit (PICU) (late-PN), as compared with initiating supplemental PN early (early-PN), improved PICU outcomes (1) and improved neurocognitive development assessed 2 years later (2). The latter was explained by avoiding early-PN induced adversely altered DNA-methylation of 37 CpG sites (3). As a large number of patients were younger than 1 year of age at randomization and given that assessment of most neurocognitive domains is only possible from 4 years of age onwards, we performed a 4-year follow-up to determine the impact of late-PN versus early-PN on physical, neurocognitive, and emotional/behavioral development. This pre-planned, 4-year follow-up study of the 1440 PEPaNIC patients and of 369 matched healthy children was blinded for treatment allocation (ClinicalTrials.gov-NCT01536275). Methods: Studied clinical outcomes included anthropometrics, health status, parent/caregiver-reported executive functions, and emotional/behavioral problems, and clinical tests for intelligence, visual-motor integration, alertness, motor coordination and memory. Univariable and multivariable linear and logistic regression analyses adjusted for risk factors assessed the impact of late-PN versus early-PN on the outcomes and investigated a potential mediation role of the adversely altered DNA-methylation of 37 CpG sites previously shown to be evoked by late-PN as compared with early-PN (3). Results: Overall, at 4 years follow-up, patients (356 late-PN patients, 328 early-PN patients) could be tested neurocognitively. They revealed worse anthropometric, health status, neurocognitive and emotional/behavioral developmental outcomes than the healthy control children. Outcomes of late-PN patients were never worse than those of early-PN patients. In contrast, late-PN patients had fewer internalizing (P=0.042) and externalizing problems (P=0.046), and fewer total emotional/behavioral problems (P=0.007) than early-PN patients, which were normalized by late-PN. Avoiding the early-PN induced adversely altered DNA-methylation status of the 37 CpG sites statistically explained its impact on the behavioral outcomes. Conclusion: Four years after randomization to late-PN or early-PN in the PICU, late-PN did not show harm, and was found to protect against emotional/behavioral problems, with altered DNA-methylation as a potential biological mediator hereof. These data further support de-implementation of PN-use early during critical illness in infants and children. (1) Fivez et al. N Eng J Med 2016 (2) Verstraete et al. Lancet Respir Med 2019 (3) Guiza et al. Lancet Respir Med 2020 (in press)

MiR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases.

MicroRNAs (miRNAs, miRs) are small noncoding RNAs that modulate gene expression by negatively regulating translation of target genes. Although the role of several miRNAs in vascular smooth muscle cells (VSMCs) has been extensively characterized, the function of miRNA-128-3p (miR-128) is still unknown. To determine if miR-128 modulates VSMC phenotype and to define the underlying mechanisms. We screened for miRNAs whose expression is modulated by an altered DNA methylation status in VSMCs, and among the hits, we selected miR-128. We found that miR-128 was expressed in various tissues, primary murine cells, and pathological murine and human vascular specimens. Through gain- and loss-of-function approaches, we determined that miR-128 affects VSMC proliferation, migration, differentiation, and contractility. The alterations of those properties were dependent upon epigenetic regulation of key VSMC differentiation genes; notably, Kruppel-like factor 4 was found to be a direct target of miR-128 and able to modulate the methylation status of the pivotal VSMC gene myosin heavy chain 11 (Myh11). Finally, in vivo lentiviral delivery of miR-128 prevented intimal hyperplasia in a mouse model of carotid restenosis without modifying vital cardiovascular parameters. miR-128 is a critical modulator of VSMCs and is regulated by epigenetic modifications upon stress. Its modulation in the context of disease could be exploited for therapeutic purposes.

Comprehensive analysis of DNA methylation and gene expression in orally tolerized T cells

T cell anergy is known to be a crucial mechanism for various types of immune tolerance, including oral tolerance. The expression of several anergy-specific genes was reportedly up-regulated in anergic T cells, and played important roles in the cells. However, how the genes were up-regulated has not been understood. In this study, we comprehensively analyzed the altered gene expression and DNA methylation status in T cells tolerized by oral antigen in vivo. Our results showed that many genes were significantly up-regulated in the orally tolerized T cells, and most of the genes found in this study have not been reported previously as anergy related genes; for example, ribosomal protein L41 (FC = 3.54E06, p = 3.70E-09: Fisher's exact test; the same applies hereinafter) and CD52 (FC = 2.18E05, p = 3.44E-06). Furthermore, we showed that the DNA methylation statuses of many genes; for example, enoyl-coenzyme A delta isomerase 3 (FC = 3.62E-01, p = 3.01E-02) and leucine zipper protein 1 (FC = 4.80E-01, p = 3.25E-02), including the ones distinctly expressed in tolerized T cells; for example, latexin (FC = 3.85E03, p = 4.06E-02 for expression; FC = 7.75E-01, p = 4.13E-01 for DNA methylation) and small nuclear ribonucleoprotein polypeptide F (FC = 3.12E04, p = 4.46E-04 for expression; FC = 8.56E-01, p = 5.15E-01 for DNA methylation), changed during tolerization, suggesting that the distinct expression of some genes was epigenetically regulated in the tolerized T cells. This study would contribute to providing a novel clue to the fine understanding of the mechanism for T cell anergy and oral tolerance.

An Altered DNA Methylation Status in the Human Umbilical Cord Is Correlated with Maternal Exposure to Polychlorinated Biphenyls

Maternal exposure to polychlorinated biphenyls (PCBs) results in abnormal fetal development, possibly because of epigenetic alterations. However, the association between PCB levels in cord serum with fetal DNA methylation status in cord tissue is unclear. This study aims to identify alterations in DNA methylation in cord tissue potentially associated with PCB levels in cord serum from a birth cohort in Chiba, Japan (male neonates = 32, female neonates = 43). Methylation array analysis identified five sites for female neonates (cg09878117, cg06154002, cg06289566, cg12838902, cg01083397) and one site for male neonates (cg13368805) that demonstrated a change in the methylation degree. This result was validated by pyrosequencing analysis, showing that cg06154002 (tudor domain containing 9: TDRD9) in cord tissue from female neonates is significantly correlated with total PCB levels in cord serum. These results indicate that exposure to PCBs may alter TDRD9 methylation levels, although this hypothesis requires further validation using data obtained from female neonates. However, since the present cohort is small, further studies with larger cohorts are required to obtain more data on the effects of PCB exposure and to identify corresponding biomarkers.

PF536 THE EFFECT OF 5‐AZACITIDINE TREATMENT ON THE OF BONE MARROW MESENCHYMAL STEM TRANSCRIPTOME IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

Background:Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cells (HSC), characterized by inefficient bone marrow (BM) haemopoiesis and increased risk for leukaemic evolution. However, MDS pathogenesis is not restricted to the HSC compartment. Emerging evidence suggests a disease permitting or even a disease promoting role for the BM microenviroment (Cogle et al,2015).Mesenchymal stem cells (MSCs) are a key component of the BM niche. MDS derived MSCs (MDS‐MSCs) have been shown to hold intrinsic functional defects and an in ability to sustain normal haemopoesis (Bulycheva et al, 2015). Moreover, recent data suggest that MDS‐MSCs harbor genetic abnormalities (Blau et al,2011) and also an altered DNA methylation status (Poloni et al,2014).5‐Azacitidine (AZA), a hypomethylating agent, is widely used for MDS treatment. Our (Liapis et al,ASH 2017) and other groups have previously shown that AZA is able to partially reverse the proliferative and clonogenic deficiencies of MDS‐MSCs, whereas it is unable to affect their intrinsic genomic instability. However, knowledge of its effect on the MDS‐MSC transcriptome is still lacking.Aims:We aim to provide the first comparative analysis of the MDS‐MSC transcriptome before and after long term AZA treatment.Methods:BM samples were collected from 12 patients with high risk MDS after informed consent. Repeat harvest, after 6 cycles of AZA therapy was possible in 7 of these patients. BM samples were also collected from 7 healthy subjects undergoing orthopedic surgery. MSCs were then isolated, ex vivo expanded and re‐seeded for a total of 5 passages (P). At P2, expanded MSCs, fulfilled the minimal criteria for MSC definition (Dominici et al, 2006) and consisted of a homogenous population. Total RNA was harvested from P2 MSCs and subjected to RNA sequencing assays. We used the QuantSeq 3’ mRNA‐Seq Library Prep Kit FWD for Illumina. It utilizes the 3′prime method of sequencing only the 3′region of the RNAs and also provides quantative measurements.Results:Thus far, data analysis has been completed in 5 paired MDS‐MSC samples, before and after AZA therapy. Proliferation and clonogenicity parameters showed a marked improvement upon AZA treatment.410 genes were shown to have an over 2∗log2 change in their expression. Of these genes, 66 were found to be differently regulated with statistical significance (p &lt; 0.05), in spite of the low sample count.Surprisingly, despite AZA's function as a transcriptional promoter, long termtreatment conferred MSC gene downregulation rather than upregulation: 228 genes exhibited transcriptional suppression. Significantly downregulated genes included members of proliferative signaling cascades, among which,effectors of PI3k‐Akt and Wnt signaling. Other affected cellular processes were those of apoptosis, extracellular matrix synthesis and stromal adherence. Notable mentions of downregulated genes are those of CCND2, encoding for cyclin D2 (‐3.317∗log2, q = 0.013), the pro‐apoptotic protein BAX (‐3.05∗log2, q = 0.013) and FRZ2 (‐2.27∗log2, q = 0.013) a mediator for Wnt signaling.182 genes were shown to be over 2∗log2 fold upregulated. Statistically significant difference was found in just 3 of them and thus, no safe conclusion can be drawn. However, analysis of the remaining MDS‐MSC and normal MSC RNA samples is still in process.Summary/Conclusion:Transcriptome analysis reveals that long term AZA treatment is able to restore MSC stemness. The inhibition of both the mitotic and apoptotic processes contributes to stem cell quiescence and longevity.

Maternal folic acid depletion during early pregnancy increases sensitivity to squamous tumor formation in the offspring in mice.

Gestational nutrition is widely recognized to affect an offspring's future risk of lifestyle-related diseases, suggesting the involvement of epigenetic mechanisms. As folic acid (FA) is a nutrient essential for modulating DNA methylation, we sought to determine how maternal FA intake during early pregnancy might influence tumor sensitivity in an offspring. Dams were maintained on a FA-depleted (FA(-)) or normal (2 mg FA/kg; FA(+)) diet from 2 to 3 days before mating to 7 days post-conception, and their offspring were challenged with chemical tumorigenesis using 7,12-dimethylbenz[a)anthracene and phorbol 12-myristate 13-acetate for skin and 4-nitroquinoline N-oxide for tongue. In both squamous tissues, tumorigenesis was more progressive in the offspring from FA(-) than FA(+) dams. Notably, in the skin of FA(-) offspring, the expression and activity of cylindromatosis (Cyld) were decreased due to the altered DNA methylation status in its promoter region, which contributed to increased tumorigenesis coupled with inflammation in the FA(-) offspring. Thus, we conclude that maternal FA insufficiency during early pregnancy is able to promote neoplasm progression in the offspring through modulating DNA methylation, such as Cyld. Moreover, we propose, for the first time, "innate" utero nutrition as the third cause of tumorigenesis besides the known causes-hereditary predisposition and acquired environmental factors.

Bisphenol A induced abnormal DNA methylation of ovarian steroidogenic genes in rare minnow Gobiocypris rarus

Bisphenol A (BPA), an ubiquitous environmental endocrine disruptor chemical, disturbs the mRNA expressions of steroidogenic genes and subsequently steroid hormone synthesis in mammals and aquatic species. However, the underlying regulation mechanisms are barely understood, especially in fish. To explore the regulation mechanism, we exposed female rare minnow Gobiocypris rarus (G. rarus) to BPA at a nominal concentration of 15 μg/L for 7 and 14 days in the present study. Results showed significant increase of gonad somatic index (GSI) and serum estradiol (E2) levels in response to BPA at day 14. The 7-day BPA exposure notably repressed the expression of two ovarian steroidogenic genes (star and hsd11b2) and suppressed their capacity of estrogen response elements (ERE) to recruit estrogen receptor (ER), while the 14-day BPA treatment remarkably induced transcript of hsd3b and enhanced the capacity of ERE to recruitment ER in ovaries. Furthermore, the 7-day BPA exposure caused DNA hypermethylation of star (CpGs: −742 bp and −719 bp) and hsd11b2 (CpG: −1788 bp). However, 14-day BPA exposure resulted in DNA hypomethylation of hsd3b (CpG: −181 bp). Correlation analysis revealed that the DNA methylation levels at specific CpGs in star, hsd3b and hsd11b2 were significantly correlated to their mRNA levels and ER-EREs interactions. These findings suggest that the disturbed steroidogenesis and the transcripts of ovarian steroidogenic genes might attribute to the altered DNA methylation status of these ovarian steroidogenic genes in response to BPA.

Multifactorial analysis of the stochastic epigenetic variability in cord blood confirmed an impact of common behavioral and environmental factors but not of in vitro conception

BackgroundAn increased incidence of imprint-associated disorders has been reported in babies born from assisted reproductive technology (ART). However, previous studies supporting an association between ART and an altered DNA methylation status of the conceived babies have been often conducted on a limited number of methylation sites and without correction for critical potential confounders. Moreover, all the previous studies focused on the identification of methylation changes shared among subjects while an evaluation of stochastic differences has never been conducted. This study aims to evaluate the effect of ART and other common behavioral or environmental factors associated with pregnancy on stochastic epigenetic variability using a multivariate approach.ResultsDNA methylation levels of cord blood from 23 in vitro and 41 naturally conceived children were analyzed using the Infinium HumanMethylation450 BeadChips. After multiple testing correction, no statistically significant difference emerged in the number of cord blood stochastic epigenetic variations or in the methylation levels between in vitro- and in vivo-conceived babies. Conversely, four multiple factor analysis dimensions summarizing common phenotypic, behavioral, or environmental factors (cord blood cell composition, pre or post conception supplementation of folates, birth percentiles, gestational age, cesarean section, pre-gestational mother’s weight, parents’ BMI and obesity status, presence of adverse pregnancy outcomes, mother’s smoking status, and season of birth) were significantly associated with stochastic epigenetic variability. The stochastic epigenetic variation analysis allowed the identification of a rare imprinting defect in the locus GNAS in one of the babies belonging to the control population, which would not have emerged using a classical case-control association analysis.ConclusionsWe confirmed the effect of several common behavioral or environmental factors on the epigenome of newborns and described for the first time an epigenetic effect related to season of birth. Children born after ART did not appear to have an increased risk of genome-wide changes in DNA methylation either at specific loci or randomly scattered throughout the genome. The inability to identify differences between cases and controls suggests that the number of stochastic epigenetic variations potentially induced by ART was not greater than that naturally produced in response to maternal behavior or other common environmental factors.

Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm.

DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A) transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation changes induced by hDNMT3A expression were intergenerationally inherited by offspring without transmission of the transgene, which provided evidence for the transmission of active endogenous-factors-induced epigenetic variations.