Hydroxymethylated DNA Research Articles

Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice.

Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects. Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25mg DEHP/kg of food (approximately 5mg DEHP/kg body weight) or 32ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5-7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15. Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood. Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects.

High-Dose Vitamin C Prevents Secondary Brain Damage After Stroke via Epigenetic Reprogramming of Neuroprotective Genes.

Vitamin C has recently been identified as an epigenetic regulator by activating ten-eleven translocases (TETs), enzymes involved in generating DNA hydroxymethylcytosine (5hmC). Currently, we investigated whether high-dose vitamin C promotes neuroprotection through epigenetic modulation of 5hmC, if there are sex-specific differences in outcome, and the therapeutic potential of vitamin C in stroke-related comorbidities in adult mice. Post-stroke treatment with ascorbate (reduced form), but not dehydroascorbate (oxidized form), increased TET3 activity and 5hmC levels and reduced infarct following focal ischemia. Hydroxymethylation DNA immunoprecipitation sequencing showed that ascorbate increased 5hmC across the genome and specifically in promoters of several stroke pathophysiology-related genes, particularly anti-inflammatory genes. Ascorbate also decreased markers of oxidative stress, mitochondrial fragmentation, and apoptosis in cortical peri-infarct neurons and promoted motor and cognitive functional recovery in both sexes via TET3. Furthermore, post-stroke ascorbate treatment reduced infarct volume and improved motor function recovery in aged, hypertensive and diabetic male and female mice. Delayed ascorbate treatment at 6h of reperfusion was still effective at reducing infarct volume and motor impairments in adult mice. Collectively, this study shows that post-stroke treatment with high-dose ascorbate protects the brain through epigenetic reprogramming and may function as a robust therapeutic against stroke injury.

Acute high folic acid treatment in SH-SY5Y cells with and without MTHFR function leads to gene expression changes in epigenetic modifying enzymes, changes in epigenetic marks, and changes in dendritic spine densities.

Epigenetics are known to be involved in various disorders, including neurobiological disorders like autism. Dietary factors such as folic acid can affect epigenetic marks using methylenetetrahydrofolate reductase (MTHFR) to metabolize folic acid to a one-carbon methyl group. As MTHFR mutations are frequent, it is curious as to whether excess folic acid, with or without functioning MTHFR, could affect gene expression, epigenetics, and neuromorphology. Here, we investigated gene expression and activity of epigenetic modifying enzymes, genome-wide DNA methylation, histone 3 modifications, and dendritic spine densities in SH-SY5Y cells with or without a knockdown of MTHFR and with or without an excess of folic acid. We found alterations to gene expression of epigenetic modifying enzymes, including those associated with disorders like autism. Grouping the epigenetic modifying enzymes by function indicated that gene expression was widely affected for genes that code for enzymes affecting DNA methylation, histone acetylation, histone methylation, histone phosphorylation, and histone ubiquitination when excess folic acid treatment occurred with or without the knockdown of MTHFR. MTHFR was significantly reduced upon excess folic acid treatment whether MTHFR was knocked-down or not. Further, methyl-CpG binding protein 2 expression was significantly decreased with excess folic acid treatment with and without proper MTHFR expression. Global DNA methylation decreased due to the knockdown alone while global hydroxymethylated DNA increased due to the knockdown alone. TET2 expression significantly increased with the MTHFR knockdown alone. Excess folic acid alone induced a decrease in TET3 expression. Excess folic acid induced an increase in dendritic spines without the MTHFR knockdown, but folic acid induced a decrease in dendritic spines when MTHFR was knocked-down. The knockdown alone also increased the dendritic spines significantly. Histone 3 acetylation at lysine 18 was significantly increased when excess folic acid was applied to cells with the MTHFR knockdown, as was histone 3 phosphorylation at serine 10. Broadly, our results indicate that excess folic acid, even with functioning MTHFR, could have detrimental effects on cells.

Aberrant hydroxymethylation of ANGPTL4 is associated with selective intrauterine growth restriction in monochorionic twin pregnancies

ABSTRACT Selective intrauterine growth restriction (sIUGR) is a severe complication in monochorionic (MC) twin pregnancies, and it carries increased risks of poor prognosis. Current data suggest that vascular anastomoses and unequal placental sharing may be the key contributor to discordant foetal growth. While MC twins derive from a single zygote and have almost identical genetic information, the precise mechanisms remain unknown. DNA hydroxymethylation is a newly discovered epigenetic feature associated with gene regulation and modification. Here, we investigate discordant hydroxymethylation patterns between two placental shares of sIUGR and analyse the potential role of aberrant hydroxymethylation of angiopoietin-like 4 (ANGPTL4) in placental dysplasia. Hydroxymethylation DNA immunoprecipitation (hMeDIP)-chip and mRNA sequencing were performed to identify hydroxymethylation-associated genes. Real-time qPCR, western blotting, and immunohistochemistry were used to confirm ANGPTL4 expression. The mechanisms regulating ANGPTL4 were investigated by cell migration assay, invasion assay, viability assay, and apoptotic ratio assays, western blotting and hMeDIP-qPCR. Decreased ANGPTL4 was detected in the smaller placental shares of sIUGR. ANGPTL4 knockdown suppressed trophoblast invasiveness and migration, which possibly occurred through hypoxia inducible factor 1α (HIF-1α) and HIF-1 signalling pathway. Hypoxia leads to aberrant expression of ANGPTL4 and HIF-1α, positively correlated with their aberrant hydroxymethylation levels in promoter regions. Aberrant hydroxymethylation of ANGPTL4 may contribute to placental impairment by the HIF-1 signalling pathway in smaller placental shares of sIUGR.

Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping.

EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution.

Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients.

Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.

Longitudinal Effects of Developmental Bisphenol A Exposure on Epigenome-Wide DNA Hydroxymethylation at Imprinted Loci in Mouse Blood.

Background:Epigenetic machinery plays an important role in genomic imprinting, a developmental process that establishes parent-of-origin–specific monoallelic gene expression. Although a number of studies have investigated the role of 5-methylcytosine in imprinting control, the contribution of 5-hydroxymethylcytosine (5-hmC) to this epigenetic phenomenon remains unclear.Objectives:Using matched mouse blood samples (from mice at 2, 4, and 10 months of age), our objective was to examine the effects of perinatal bisphenol A (BPA) exposure ( diet) on longitudinal 5-hmC patterns at imprinted regions. We also aimed to test the hypothesis that 5-hmC would show defined patterns at imprinted genes that persist across the life course.Methods:Genome-wide 5-hmC levels were measured using hydroxymethylated DNA immunoprecipitation sequencing (HMeDIP-seq). Modeling of differential hydroxymethylation by BPA exposure was performed using a pipeline of bioinformatics tools, including the csaw R package.Results:Based on BPA exposure, we identified 5,950 differentially hydroxymethylated regions (DHMRs), including 12 DHMRs that were annotated to murine imprinted genes—Gnas, Grb10, Plagl1, Klf14, Pde10a, Snrpn, Airn, Cmah, Ppp1r9a, Kcnq1, Phactr2, and Pde4d. When visualized, these imprinted gene DHMRs showed clear, consistent patterns of differential 5-hmC by developmental BPA exposure that persisted throughout adulthood.Conclusions:These data show long-term establishment of 5-hmC marks at imprinted loci during development. Further, the effect of perinatal BPA exposure on 5-hmC at specific imprinted loci indicates that developmental exposure to environmental toxicants may alter long-term imprinted gene regulation via an epigenetic mechanism. https://doi.org/10.1289/EHP3441

FAM20C could be targeted by TET1 to promote odontoblastic differentiation potential of human dental pulp cells.

Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation. The expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter. FAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction. TET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression.

Global DNA methylation analysis reveals miR-214-3p contributes to cisplatin resistance in pediatric intracranial nongerminomatous malignant germ cell tumors.

Pediatric central nervous system germ cell tumors (CNSGCTs) are rare and heterogeneous neoplasms, which can be divided into germinomas and nongerminomatous germ cell tumors (NGGCTs). NGGCTs are further subdivided into mature teratomas and nongerminomatous malignant GCTs (NGMGCTs). Clinical outcomes suggest that NGMGCTs have poor prognosis and survival and that they require more extensive radiotherapy and adjuvant chemotherapy. However, the mechanisms underlying this difference are still unclear. DNA methylation alteration is generally acknowledged to cause therapeutic resistance in cancers. We hypothesized that the pediatric NGMGCTs exhibit a different genome-wide DNA methylation pattern, which is involved in the mechanism of its therapeutic resistance. We performed methylation and hydroxymethylation DNA immunoprecipitation sequencing, mRNA expression microarray, and small RNA sequencing (smRNA-seq) to determine methylation-regulated genes, including microRNAs (miRNAs). The expression levels of 97 genes and 8 miRNAs were correlated with promoter DNA methylation and hydroxymethylation status, such as the miR-199/-214 cluster, and treatment with DNA demethylating agent 5-aza-2'-deoxycytidine elevated its expression level. Furthermore, smRNA-seq analysis showed 27 novel miRNA candidates with differential expression between germinomas and NGMGCTs. Overexpresssion of miR-214-3p in NCCIT cells leads to reduced expression of the pro-apoptotic protein BCL2-like 11 and induces cisplatin resistance. We interrogated the differential DNA methylation patterns between germinomas and NGMGCTs and proposed a mechanism for chemoresistance in NGMGCTs. In addition, our sequencing data provide a roadmap for further pediatric CNSGCT research and potential targets for the development of new therapeutic strategies.

Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus.

Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state.IMPORTANCE Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.

Emerging Role of One-Carbon Metabolism and DNA Methylation Enrichment on δ-Containing GABAA Receptor Expression in the Cerebellum of Subjects with Alcohol Use Disorders (AUD).

BackgroundCerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic α6/δ-containing GABAA receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABAA receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.MethodsWe used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). S-adenosyl-methionine/S-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.ResultsmRNAs encoding key enzymes of 1-carbon metabolism that determine the S-adenosyl-methionine/S-adenosyl-homocysteine ratio were increased, indicating higher “methylation index” in alcohol use disorder subjects. We found that increased methylation of the promoter of the δ subunit GABAA receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in α1- or α6-containing GABAA receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the δ subunit GABAA receptor promoter may result from alcohol-induced reduction of DNA demethylation.ConclusionTogether, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABAA receptor function.

Molecular cloning of chicken TET family genes and role of chicken TET1 in erythropoiesis

Ten-eleven translocation (TET) methylcytosine dioxygenase has potential as an active eraser to regulate the genomic DNA methylation status. We herein cloned chicken TET (cTET) family genes, and confirmed their functions. Quantitative reverse-transcription PCR showed that cTET1 was strongly expressed in erythrocytes throughout development. This cTET1 expression pattern, together with the results of methylated or hydroxymethylated DNA immunoprecipitation, suggests that cTET1 contributes to demethylation around the promoter region of the definitive-type β-globin gene βΑ in erythroid cells. The knockdown of cTET1 in T2ECs chicken erythroid progenitor cells suppressed the induction of βΑ expression under differentiation conditions. These results suggest that cTET1 plays an important role in erythroid cell differentiation.

The effect of paternal methyl-group donor intake on offspring DNA methylation and birth weight.

Most nutritional studies on the development of children focus on mother-infant interactions. Maternal nutrition is critically involved in the growth and development of the fetus, but what about the father? The aim is to investigate the effects of paternal methyl-group donor intake (methionine, folate, betaine, choline) on paternal and offspring global DNA (hydroxy)methylation, offspring IGF2 DMR DNA methylation, and birth weight. Questionnaires, 7-day estimated dietary records, whole blood samples, and anthropometric measurements from 74 fathers were obtained. A total of 51 cord blood samples were collected and birth weight was obtained. DNA methylation status was measured using liquid chromatography-tandem mass spectrometry (global DNA (hydroxy)methylation) and pyrosequencing (IGF2 DMR methylation). Paternal betaine intake was positively associated with paternal global DNA hydroxymethylation (0.028% per 100 mg betaine increase, 95% CI: 0.003, 0.053, P=0.03) and cord blood global DNA methylation (0.679% per 100 mg betaine increase, 95% CI: 0.057, 1.302, P=0.03). Paternal methionine intake was positively associated with CpG1 (0.336% per 100 mg methionine increase, 95% CI: 0.103, 0.569, P=0.006), and mean CpG (0.201% per 100 mg methionine increase, 95% CI: 0.001, 0.402, P=0.049) methylation of the IGF2 DMR in cord blood. Further, a negative association between birth weight/birth weight-for-gestational age z-score and paternal betaine/methionine intake was found. In addition, a positive association between choline and birth weight/birth weight-for-gestational age z-score was also observed. Our data indicate a potential impact of paternal methyl-group donor intake on paternal global DNA hydroxymethylation, offspring global and IGF2 DMR DNA methylation, and prenatal growth.

Role of oxidative stress and DNA hydroxymethylation in the neurotoxicity of fine particulate matter

Epidemiological studies have implicated fine particulate matter (PM2.5) as a risk factor for neurodegenerative diseases and neurodevelopmental disorders. However, the underlying molecular mechanisms and the influences of different components remain largely elusive. Here, we extended our previous work to investigate the role of oxidative stress and DNA hydroxymethylation in neuronal pathology of PM2.5. We found PM2.5 and its extracts (water-soluble extracts, organic extracts and carbon core component) differentially caused cell cycle arrest, cell apoptosis and the cell proliferation inhibition in neuronal cells. These effects were mechanistically related to each other and oxidative stress, suggesting PM2.5 and toxic compounds adsorbed on the particles may cause different types of brain damages. In addition, PM2.5 and its organic extracts increased global DNA hydroxymethylation and gene-specific DNA hydroxymethylation of neuronal genes, and subsequently interfered with their mRNA expression. The impairments in neuronal progression characterized with decreased length of neurite and reduced mRNA expression of neuronal markers and synaptic markers. The blocking effects of antioxidants demonstrated the involvement of oxidative stress-mediated hydroxymethylation abnormalities in PM2.5-induced defects in neurite outgrowth and synapse formation. Our results first revealed the role of oxidative stress-mediated abnormal DNA hydroxymethylation in neuronal impairments of PM2.5, and thoroughly evaluated the neurocytotoxicity of different components.

The Mechanism Underlying the Balance Between Heat Stress Resilience and Vulnerability are Mediated by Dna Methylation and De-Methylation at a Regulatory Segment of the Dna Encoding Corticotrophin-Releasing-Hormone

Background Determining whether stress will lead to future stress-resilience or vulnerability depends on a delicate balance of a probably adjustable stress response set-point. The adjustment of this set-point is most likely effective during sensory postnatal development and involves the hypothalamus-pituitary-adrenal (HPA) axis. Here we demonstrate that heat stress during the critical period of thermal control establishment in 3-day-old chicks, renders habituated or sensitized response, a week later, depending on the ambient temperature i.e. moderate heat lead to future heat resistance while harsh temperatures lead to heat vulnerability. Furthermore, these changes are governed by epigenetic modifications, specifically DNA methylation (5-methycytosine - 5mc) and DNA hydroximethylation (5-hydroxymethylcytosine - 5hmc). Methods In this study we made use of several innovative methods, primarily: RNA and DNA purification Reverse transcription to cDNA qRT-PCR Global hydroxymethylated DNA (5-hmC) Quantification, ELISA Global DNA methylation (5mC) Quantification, ELISA Bisulfit treatment followed by ligation of DNA to plasimd and transformation to E.coli DNA sequencing of specific targets of interest from the CRH gene, from the cloned DNA Western blotting Chromatin immunoprecipitation (ChIP) Immunohistology Intracranial injection of different treatments to live 3 day old chicks. Results After concluding that CRH plays a significant role in determining either heat resilience or vulnerability response later in life, we examine epigenetic changes as a potential mechanism. Interestingly, global 5mc% and global 5hmc% changed significantly between the two different heat conditioned groups, a week after their conditioning. Resilient chicks displayed low 5mc% alongside high 5hmc%, while vulnerable chicks displayed an opposite pattern of high 5mc% and low 5hmc%. This pattern was repeated when specific CpG methylation sites along the CRH gene were evaluated, indicating these dynamic changes contribute to the differences in the expression levels of CRH, and therefore might take part in the formation of the habituation and resilience phenomena. By injecting Parp (poly ADP-ribose polymerase) inhibitor - which is required for activation of Tet (ten-eleven translocation demethylation intermediate family) activity, the 5hmc% declined, along with a decline in 5mc%. This in turn resulted in obliteration of the ambient temperature resilience showed previously by non-injected chicks, when compared to their control counterparts. Discussion Long term changes in body temperature, mRNA expression and Cort levels of chicks conditioned at different intensities of stress, define the basis of resilience versus vulnerability response later in life. Here we show that these changes are directly affected by CRH, demonstrated by injecting CRH or CRH-antisense which inverted the normal response. Furthermore, we propose that DNA methylation and de-methylation take part in this long term phenomena as indicated both by global and gene specific 5mc% and 5hmc% one week after heat conditioning. Moreover, injection of PAPR inhibitor, which is required for activation of the Tet demethylation intermediate family activity, resulted in obliteration of the ambient temperature resilience showed previously by non-injected chicks. It is highly plausible to assume that the mechanism underlying the balance between heat stress resilience and vulnerability is regulated by epigenetic adaptations.

DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.

TET2 Negatively Regulates Nestin Expression in Human Melanoma

Although melanoma is an aggressive cancer, the understanding of the virulence-conferring pathways involved remains incomplete. We have demonstrated that loss of ten-eleven translocation methylcytosine dioxygenase (TET2)-mediated 5-hydroxymethylcytosine (5-hmC) is an epigenetic driver of melanoma growth and a biomarker of clinical virulence. We also have determined that the intermediate filament protein nestin correlates with tumorigenic and invasive melanoma growth. Here we examine the relationships between these two biomarkers. Immunohistochemistry staining of nestin and 5-hmC in 53 clinically annotated primary and metastatic patient melanomas revealed a significant negative correlation. Restoration of 5-hmC, as assessed in a human melanoma cell line by introducing full-length TET2 and TET2-mutated constructs, decreased nestin gene and protein expression invitro. Genome-wide mapping using hydroxymethylated DNA immunoprecipitation sequencing disclosed significantly less 5-hmC binding in the 3' untranslated region of the nestin gene in melanoma compared to nevi, and 5-hmC binding in this region was significantly increased after TET2 overexpression in human melanoma cells invitro. Our findings provide evidence suggesting that nestin regulation is negatively controlled epigenetically by TET2 via 5-hmC binding at the 3' untranslated region of the nestin gene, providing one potential pathway for understanding melanoma growth characteristics. Studies are now indicated to further define the interplay between 5-hmC, nestin expression, and melanoma virulence.

The dynamics of DNA methylation in schizophrenia and related psychiatric disorders.

Major psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BP) with psychosis (BP+) express a complex symptomatology characterized by positive symptoms, negative symptoms, and cognitive impairment. Postmortem studies of human SZ and BP+ brains show considerable alterations in the transcriptome of a variety of cortical structures, including multiple mRNAs that are downregulated in both inhibitory GABAergic and excitatory pyramidal neurons compared with non-psychiatric subjects (NPS). Several reports show increased expression of DNA methyltransferases in telencephalic GABAergic neurons. Accumulating evidence suggests a critical role for altered DNA methylation processes in the pathogenesis of SZ and related psychiatric disorders. The establishment and maintenance of CpG site methylation is essential during central nervous system differentiation and this methylation has been implicated in synaptic plasticity, learning, and memory. Atypical hypermethylation of candidate gene promoters expressed in GABAergic neurons is associated with transcriptional downregulation of the corresponding mRNAs, including glutamic acid decarboxylase 67 (GAD67) and reelin (RELN). Recent reports indicate that the methylation status of promoter proximal CpG dinucleotides is in a dynamic balance between DNA methylation and DNA hydroxymethylation. Hydroxymethylation and subsequent DNA demethylation is more complex and involves additional proteins downstream of 5-hydroxymethylcytosine, including members of the base excision repair (BER) pathway. Recent advances in our understanding of altered CpG methylation, hydroxymethylation, and active DNA demethylation provide a framework for the identification of new targets, which may be exploited for the pharmacological intervention of the psychosis associated with SZ and possibly BP+.