A epigenética é uma subárea da Genética, na qual são estudados mecanismos que são essenciais para o adequado desenvolvimento dos mamíferos, sendo que, alterações neste estágio podem levar a vários distúrbios metabólicos, como a obesidade. Atualmente a obesidade é um grave problema de saúde pública mundial, tem origem multifatorial envolvendo tanto fatores ambientais quanto genéticos. Existem alguns genes que estão envolvidos com a obesidade, como por exemplo, o gene da pró-opiomelanocortina (POMC). O objetivo do presente projeto de pesquisa é investigar os padrões de expressão e metilação do gene Pomc na prole de ratas submetidas às dietas deficiente e suplementada com ácido fólico. Os animais utilizados foram ratos Wistar. O estudo envolveu filhotes (n=24) machos e fêmeas que foram desmamados com a mesma dieta de suas respectivas mães, sendo três grupos de tratamento, o grupo controle (2,0 mg de ácido fólico/kg de ração), o grupo deficiente (0,5 mg de ácido fólico/kg de ração) e o grupo suplementado (8 mg de ácido fólico/kg de ração). Foram coletadas amostras do núcleo arqueado do hipotálamo, a partir das quais foram extraídos DNA, RNA e proteínas utilizando kits comerciais seguindo o protocolo do fabricante. Com o DNA foi realizada a análise do padrão de metilação. O mRNA foi utilizado para a análise da expressão gênica, por PCR em tempo real, pelo sistema TaqMan® (Life Technologies(TM)). O estudo de proteômica foi realizado por Western blotting. De modo geral, observou-se que o peso corpóreo dos filhotes machos não apresentou diferença estatística entre os grupos. O consumo de ração do grupo deficiente em ácido fólico foi estatisticamente (p = 0,03) maior do que o grupo controle. Em relação aos filhotes fêmeas observou-se que o peso corpóreo do grupo suplementado foi estatisticamente (p = 0,01) maior do que o grupo controle, e referente ao consumo de ração, não houve diferença estatística significativa entre os grupos de tratamento. As análises de peso cerebral, expressão gênica, metilação e expressão proteica de Pomc não apresentaram diferenças estatísticas significativas entre os grupos de tratamento de ambos os sexos. Conclui-se que a intervenção com dietas com diferentes concentrações de ácido fólico não ocasionou alterações significativas na prole, em relação ao estudo de proteômica e aos padrões de metilação e expressão do gene Pomc. Quanto ao peso corpóreo e consumo de ração dos animais mostrou-se que a suplementação com ácido fólico durante a gestação e no pós desmame foi capaz de alterar estes dois parâmetros, com resposta divergente entre os machos e fêmeas na prole adulta. / Epigenetic mechanisms are essential for proper development in mammals, and that changes at this stage may lead to various metabolic disorders such as obesity. Currently obesity is a serious problem of public health worldwide, has a multifactorial origin involving both environmental and genetic factors. There are some genes that are involved with obesity, such as the proopiomelanocortin gene (POMC). The aim of this research project is to investigate the expression and methylation patterns of the Pomc gene in the offspring of rats subjected to deficient and supplemented diets with folic acid. Animals used were Wistar rats. The study involved males and females pups (n = 24) that were weaned at the same diet their mothers, three treatment groups, control group (2,0 mg/kg of folic acid), deficient group (0,5 mg/kg of folic acid) and the supplemented group (8,0 mg/kg of folic acid). The arcuate nucleus of the hypothalamus tissue were collected, from which was extracted DNA, RNA and proteins using commercially available kits following the manufacturer\'s protocol. The DNA methylation pattern analysis was performed. The mRNA was used for the analysis of gene expression by real time PCR, the TaqMan (Life Technologies (TM)) system. The proteomic study was carried out by Western blotting. In general, we found that the body weight of the male offspring showed no statistical difference between the groups. The feed intake of folic acid deficient group was statistically (p = 0.03) higher than the control group. In relation to female offspring was observed that the body weight of the supplemented group was statistically (p = 0.01) higher than the control group, and related to feed intake, there was no statistically significant difference between the treatment groups. The analysis of brain weight, gene expression, methylation and protein expression of Pomc no significant statistical differences among treatment groups of both sexes. Concluded that the intervention diets with different folic acid concentrations did not cause significant changes in the offspring compared to the study of proteomics and methylation and expression patterns of the Pomc gene. As for the body weight and feed consumption of animals it showed that supplementation with folic acid during pregnancy and post weaning was able to alter these two parameters with differing response between males and females in the adult offspring.

The p300 transcriptional coactivator has been implicated in the development of a large number of malignancies; however, the precise mechanism of p300-associated tumorigenesis remains unclear. Here, we demonstrate the functional impact of p300 in human melanomas using both genetic and chemical approach. Depletion of p300 in human melanoma cells was associated with cellular growth arrest and senescence. Microarray analysis identified the Microphthalmia-associated transcription factor (MITF), a critical lineage-specific transcription factor in melanocytes and melanomas, as a major downstream target of p300 in human melanoma. Ectopic expression of MITF in p300-depleted melanoma cells allowed rescue of the p300-silencing phenotype, suggesting a critical regulatory axis involving p300 and MITF. Chromatin immunoprecipitation studies revealed direct regulation of MITF transcription through p300 acetylation of proximal regulatory domains. Critically, we identified that Forkhead Box M1 (FOXM1), a potent pro-proliferation transcription factor, is a target of the p300-MITF signaling axis. Further evaluation of p300 regulation of melanoma cell growth was performed using a highly selective p300/CBP HAT inhibitor, 228-1. Inhibition of p300/CBP histone acetyltransferase (HAT) activity was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion. Together, these data support the role of p300 as a promising therapeutic target in human melanoma and suggest particular therapeutic efficacy of small molecule inhibitors of p300 HAT activity in tumors expressing high levels of MITF. / 2018-12-14T00:00:00Z

Mastitis represents one of the major economical and animal welfare concerns within the dairy industry. Animals affected with this disease can experience a range of clinical symptoms from mild discomfort and swelling of the udder to a severe systemic inflammatory response that could result in the death of the animal. This range of responses is due to differences in pathogen, environment, and inter-animal differences in their innate immune response. A dermal fibroblast model was used to predict the magnitude of an animal's innate immune response towards an intra-mammary S. aureus challenge. Animals whose fibroblasts exhibited a low response phenotype, characterized by lower levels of IL-8 following in vitro immune stimulation, suffered less mammary tissue damage and a less severe reduction in milk quality following the in vivo S. aureus challenge as compared to animals classified as high responders. Furthermore, the heightened inflammatory response of the high responders offered no advantage in bacterial clearance. For a S. aureus infection, the lower response phenotype is preferred. To further explore inter-animal variation in the innate immune response, fibroblast cultures were established and challenged with LPS from two breeds of cattle, Holsteins, a dairy breed and Angus, a beef breed. Cultures from Holstein animals exhibited a higher responding phenotype than cultures from Angus animals. As these two breeds undergo selection for different traits and are reared differently as calves, whole transcriptome analysis (RNA-Seq) and DNA methylation analysis (Methylated CpG Island Recovery Assay; MIRA-Seq) of their fibroblasts was completed to examine the genetic and epigenetic basis for the contrasting responses. RNA-Seq revealed several immune associated genes that were expressed at higher levels in Holstein cultures compared to Angus cultures, including TLR4, IL-8, CCL5, and TNF-α, both basally and following LPS exposure. Although MIRA-Seq analysis revealed 49 regions with differential methylation between the Holstein and Angus cultures, overall, the methylation of the fibroblast genome was similar between these breeds. A combination of genetic and epigenetic factors seems to contribute to the breed-dependent differences observed between Holstein and Angus fibroblasts. Early life exposure to bacterial compounds or inflammatory mediators can have long-term effects on the magnitude of an animal's innate immune response, and may contribute to inter-animal variation in this response. To determine if an early life exposure to LPS would modify the response to a subsequent LPS challenge in dairy animals, neonatal Holstein calves were treated with LPS or saline at 7 days of age and subsequently challenged with LPS 25 days later. Calves that received LPS at 7 days of age had greatly elevated levels of plasma IL-6 and TNF-α compared to calves that received saline, indicating a substantial inflammatory response. However, following the subsequent LPS challenge completed on all calves, there were no differences in plasma IL-6 and TNF-α between the LPS- and saline- treated calves. Alternative exposure strategies in calves may generate the long-term effects observed in other model systems. There is a wide range in the responses observed in the innate immune response of the bovine. Animals with a lower innate immune response effectively clear the infection, but avoid the collateral tissue damage from excessive inflammation. Therefore, it seems that a reduced innate immune response would be more beneficial to the dairy cow.

A produção in vitro de embriões (PIVE) vem se destacado como biotecnologia reprodutiva nos rebanhos bovinos, porém ainda com algumas limitações. A diferença de fertilidade entre touros é uma delas e dentre os atributos espermáticos que podem impactar na PIVE, pouco se sabe sobre a composição do núcleo espermático. Sendo assim, este trabalho teve como objetivo principal identificar quais aspectos nucleares do espermatozoide caracterizam a diferença nos índices de PIVE. Para tanto, o estudo foi dividido em 3 experimentos nos quais foi utilizado o banco de dados da empresa In Vitro Brasil para seleção de touros de alta (AF) e baixa (BF) taxa de desenvolvimento embrionário (taxa de blastocisto/taxa de clivados; n=5 touros/grupo). No primeiro experimento, foi realizado a avaliação da cromatina para susceptibilidade ao desafio ácido (SCSA modificado), deficiência de protaminação (CMA3) e fragmentação do DNA (COMETA). Apenas a deficiência de protaminação apresentou diferença entre os grupos, entretanto a porcentagem de células positivas para o CMA3 foi baixa, não sendo possível atribuir a esta alteração as diferenças de fertilidade encontradas. No segundo experimento, foi realizada a avaliação de proteômica do núcleo espermático destes touros. Para tanto, as amostras foram incubadas com detergente CTAB para remoção de parte da membrana e cauda, extração das proteínas, digestão com tripsina e análise por espectrometria de massas. A quantificação das proteínas foi avaliada pelo índice iBAQ obtido após a identificação dos peptídeos e a média deste índice comparado entre os grupos para identificação das proteínas diferencialmente expressas. Foram identificadas 196 proteínas, 21 diferencialmente expressas, sendo 17 hiperexpressas no grupo AF e 4 hiperexpressas no grupo BF. Dentre elas, proteínas relacionadas ao processo de espermatogênese, fecundação e motilidade. No terceiro experimento, com objetivo de identificar as marcações epigenéticas relacionadas com a diferença de fertilidade in vitro, foi realizado a avaliação das estruturas de nucleossomos utilizando a enzima MNase para posteriormente utilização na técnica de imunoprecipitação das marcações de histona. Entretanto, não foi identificado a presença de nucleossomos no DNA espermático bovino, inviabilizando análises posteriores. Sendo assim, a avalição da metilação de DNA foi escolhida como marcação epigenética para ser estudada. Neste caso, foi utilizada a técnica de RRBS (reduced representation of bisulfite sequencing) para identificar as citosinas diferencialmente metiladas (CDM) entre os grupos AF e BF. Foram identificadas 440 CDM, sendo 327 hipometiladas e 113 hipermetiladas no grupo AF. Além disto, 184 genes contendo estas CDM foram identificados, entretanto nenhum processo biológico foi enriquecido por estes genes. Foi possível concluir com este trabalho que a fertilidade in vitro pode ser considerada de caráter multifatorial, sendo que alterações pequenas de integridade de cromatina não afetam diretamente, já proteínas relacionadas a espermatogênese, motilidade e fecundação podem ter relação com o fenótipo fertilidade estudado. Dentre as marcações epigenética estudadas, as estruturas de nucleossomo pareceram estar ausentes no espermatozoide bovino, e a metilação de DNA não apresenta impacto significativo no perfil de fertilidade. / In vitro embryo production (IVP) has been highlighted as reproductive biotechnique in bovine herds, but still with some limitations. The fertility difference between bulls is one of them and among the sperm attributes that can impact on IVP, little is known about the composition of the bovine sperm nucleus. Therefore, this work had as main goal to identify which sperm nuclear aspects characterize the difference in the IVP index. Then, the study was divided in 3 experiments in which the database of the company In Vitro Brasil was used for selection of bulls with high (HF) and low (LF) embryo development rate (blastocyst rate/cleavage rate; n=5 bulls/group). In the first experiment, chromatin analysis of susceptibility to acid denaturation (modified SCSA), protamine deficiency (CMA3) and DNA fragmentation (COMET assay) were performed. Only protamine deficiency presented difference between groups, however the percentage of CMA3 positive cells was low and it was not possible to attribute to this alteration the fertility difference between groups. In the second experiment, proteomic analysis of the sperm nucleus of these bulls was performed. For that, samples were incubated with CTAB detergent to remove part of the membrane and tail, submitted to protein extraction, trypsin digestion and mass spectrometry analysis. The amount of the proteins was evaluated by the iBAQ index obtained after the identification of peptides and the average of this index compared to identify the differentially expressed proteins. In this experiment, 196 proteins were identified, 21 differentially expressed, being 17 overexpressed in the HF and 4 overexpressed in LF group. Among them, proteins related to the spermatogenesis, fertilization and motility. In the third experiment, in order to identify the epigenetic marks related to in vitro fertility differences it was performed the evaluation of the nucleosome structures using the MNase enzyme to later utilization of immunoprecipitation of the histone marks technique. However, the presence of nucleosomes in bovine sperm DNA was not identified, unfeasible further analysis. Thus, the evaluation of DNA methylation was chosen as epigenetic mark to be studied. In this case, the RRBS (reduced representation of bisulfite sequencing) technique was used to identify the differentially methylated cytosines (DMC) between the two groups. In this experiment, it was identified 440 DMC, being 327 hypomethylated and 113 hypermethylated in HF group. In addition, 184 genes containing these DMCs were identified, however no biological process enrichment was identified with these genes. In conclusion, this study indicated that in vitro fertility characterization can be multifactorial; small changes in chromatin integrity do not directly affect, but proteins related to spermatogenesis, motility and fertilization may be related to the fertility phenotype studied. Among the epigenetic marks studied, nucleosome structures were absent in bovine spermatozoa, and DNA methylation did not have a significant impact on the fertility profile.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

There is a genome in a cell, as everyone knows, but there is also an epigenome. The epigenome regulates the transcription of the underlying genome. In the last decade, it was discovered that the epigenome state and its regulation are important for differentiation and development. Correlation studies with aging samples had led to the hypothesis that misregulation of the epigenome causes aging and cancer. Furthermore, diseases were identified which are caused by errors in the epigenome state and its regulation. Identification of erroneous epigenome states and misregulation requires the prior knowledge of the common state. Several studies aim at measuring epigenome states in different organisms and cell types and thus, provide a huge amount of data. In this dissertation, a pipeline is developed to analyze and characterize histone modifications with respect to different cell types. Application of this pipeline is shown for a published data set of mouse consisting of data for H3K4me3, H3K27me3, and H3K9me3 measured in embryonic stem cells, embryonic fibroblasts and neuronal progenitors. Furthermore, methods for the detection of the epigenetic patterns are presented in this dissertation. Therefore, a segmentation method is developed to segment the genome guided by the data sets. Based on this segmentation, the epigenome states as well as epigenetic variation can be studied. Different visualization methods are developed to highlight the epigenetic patterns in the segmentation data. Application of the segmentation AND visualization methods to the mouse data set had resulted in not only colorful squares but also in biological conclusions! It demonstrate the power of the developed methods. Although the studied data set in this dissertation contains only ordinary tissue cells, the methods are not restricted to study the reference epigenome state. Comparison of normal and disease cells as well as comparison with aged cells are possible with all of the methods. Finally, the methods are compared based on the obtained results. It shows that all methods highlight different aspects of the data. Thus, applying all methods to the same data sets, deep insights into the epigenome in murine embryonic stem cells, embryonic fibroblasts and neuronal progenitor cells are gained. For example, it had been found that several mechanisms exist setting H3K4me3 marks. Furthermore, not all mechanisms are found in all cell types. Strong evidence had been found that catalysis of H3K4me3 and H3K27me3 is coupled.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Traditionally, autosomal genes, when expressed, are assumed to express both alleles equally. Exceptions to this tenet include genes for which a specific genotypic polymorphism controls expression level, as well as genes for which monoallelic expression is epigenetically dictated. In this work, we discovered and characterized allele-specific gene expression in a variety of tissues using multiple techniques. We used cell lines and fresh tissue from reciprocal crosses of F1 heterozygous mice and the homozygous parental strains. We relied on a variety of high-throughput genomic techniques including RNA-Seq and DNA SNP-arrays to examine multiple types of allele-specific expression. We searched for novel examples of random autosomal monoallelic expression (RMAE) by using DNA SNP-arrays and cDNA from lymphoblast and fibroblast clonal cell lines of heterozygous mice. We found that of the approximately 1,350 autosomal genes we assessed, over 10% showed evidence of RMAE in at least one cell type. This allele-specific expression was stable over long periods in cell culture and encompassed a variety of gene types, some of which also exhibit RMAE in human clonal lines. Additionally, for a subset of RMAE genes, there seemed to be preferential inactivation of one allele; this monoallelic expression was still considered random, as from clone to clone the gene could be expressed either monoallelically or biallelically. In a second set of experiments we developed an analysis pipeline to examine RNASeq data for allele-specific expression. Using a pilot data set, we assessed the murine liver for parent-of-origin monoallelic expression, examining both known and candidate novel imprinted genes. We observed imprinted monoallelic expression in the adult liver for some, but not all, imprinted genes that have been reported in studies of embryonic tissue. The results suggest that there are few, if any, novel imprinted genes to be discovered in the mouse liver. This pilot data set also allowed examination of the genetic basis of allele specific gene expression. In keeping with recent reports, we found evidence for genetically based allele-specific expression, ranging from mild to greater than 4-fold allelic imbalance. We examined the extent to which this allelic imbalance correlated with total expression levels in the parental strains.

Valproic acid (2-propylpentanoic acid, VPA) is a clinically used anti-epileptic drug and an effective mood stabilizer. VPA is also a histone deacetylase inhibitor and can induce embryonic malformations in both humans and mice. The mechanism(s) of VPA-induced teratogenicity are not well characterized. The objectives of my study were three fold, to: (i) investigate the effect of VPA on mouse embryonic development, (ii) characterize the putative mechanism(s) of VPA-induced teratogenicity and, (iii) investigate VPA associated epigenetic regulation of Hoxa2 gene in cell lines and in developing embryos. Whole mouse embryo cultures were treated with VPA at doses of 0, 50 (0.35 mM), 100 (0.70 mM), 200 (1.4 mM), and 400 µg/mL (2.8 mM), encompassing the therapeutic range of 0.35 mM to 0.70 mM. Van Maele-Fabry’s morphologic scoring system was used to quantitatively assess embryonic organ differentiation and development. Hoxa2 gene expression was measured by quantitative real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). To assess epigenetic changes on the Hoxa2 gene promoter, DNA methylation was determined by bisulfite (BSP) sequencing and pyrosequencing. Histone “bivalent domains” H3K4me3 (histone 3 lysine 4 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation) associated with gene activation repression, respectively, analyzed qChIP-PCR (quantitative chromatin immunoprecipitation-PCR). Telomere length and telomerase activity were analyzed in mouse embryos and in NIH3T3 cell line treated with VPA. Results indicate significantly increased incidence of dysmorphogenesis in embryos (11.8%, 35.3%, 47.0% and 88.3%) exposed to increasing doses of VPA (0.35 mM, 0.70 mM, 1.4 mM and 2.8 mM respectively). Van Maele-Fabry’s quantitative differentiation assessment of developing embryos demonstrated a significantly lower score for the circulation system, central nervous system, craniofacial development and limb development in VPA treated embryos (0.35 mM to 2.8 mM) compared to the untreated control group. Glutathione homeostasis was altered as indicated by decreased total glutathione content and increased GSSG/GSH ratio in all VPA treatment groups. In addition, a dose-dependent inhibition of Hoxa2 gene expression was observed in embryos and in the NIH3T3 cell line exposed to VPA. Pre-treatment with ascorbic acid [1000 µg/mL (5 mM)] restored glutathione level and normalized Hoxa2 gene expression in embryos exposed to VPA. DNA methylation status was characterized on the Hoxa2 gene promoter at the three CpG islands; CpG island 1 (-277 to -620 bp), CpG island 2 (-919 to -1133 bp), and CpG island 3 (-1176 to -1301 bp) in the two cells lines (NIH3T3 and EG7) and in developing embryos. CpG sites remained unmethylated on the Hoxa2 gene promoter in the NIH3T3 cell line which expresses the Hoxa2 gene, whereas these same CpG sites were methylated in EG7 cells that did not express Hoxa2. CpG island 1 is closest to Hoxa2 transcription start site and its methylation status was most affected. In developing embryos, CpG island 1 was found to be highly methylated at E6.5 when Hoxa2 is not expressed, whereas the methylation status of CpG sites on the CpG island 1 declined between E8.5 and E10.5 when Hoxa2 expression is present. VPA induced methylation of several CpG sites on CpG island 1 in NIH3T3 cell line and in E10.5 embryos when Hoxa2 expression was down regulated following VPA exposure. In addition, embryos and the NIH3T3 cell line treated with VPA impacted the “bivalent domains” resulting in increased H3K27me3 enrichment and decreased H3K4me3 enrichment on Hoxa2 promoter. Pre-treatment with ascorbic acid normalized Hoxa2 expression and histone bivalent domain changes and prevented increased DNA methylation following VPA exposure. Moreover, the telomerase activity and telomere length were both impacted by changes in glutathione redox potential induced by VPA. Oxidative stress following VPA treatment reduced telomerase activity and accelerated telomere shortening. These results are the first to demonstrate: (i) a correlation between VPA dose and total morphologic score in the developing mouse embryos. VPA impacted embryonic tissue differentiation and neural system development in the dose range of 0.35 mM to 2.8 mM; (ii) VPA altered glutathione homeostasis in cultured mouse embryos and inhibited Hoxa2 gene expression; (iii) Histone bivalent domains of H3K27 and H3K4 trimethylation and DNA methylation status at the Hoxa2 gene promoter region were altered following treatment with VPA. This appears to be the epigenetic event in transcriptional silencing of Hoxa2 gene expression after VPA exposure; and (iv) Ascorbic acid normalizes glutathione homeostasis, H3K27 and H3K4 trimethylation and DNA methylation status, restoring Hoxa2 gene expression following VPA exposure. Taken together our results show VPA- induced altered glutathione homeostasis, telomere shortening and telomerase dysfunction, and an inhibition of Hoxa2 gene expression leads to developmental abnormalities. Exposure to ascorbic acid had a protective effect on developing embryos exposed to VPA.