Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

BACKGROUND: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. OBJECTIVE: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. METHODS: Female mice were exposed to human relevant doses of either Pb (32 ppm) via drinking water or DEHP (5mg/kg-day) via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. RESULTS: The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n=13 and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types (n=55 and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. CONCLUSIONS: These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

The role of epigenetics in the reproductive toxicity of environmental endocrine disruptors.

Environmental endocrine disruptors (EEDs) seriously endanger human health by interfering with the normal function of reproductive systems. In males, EEDs can affect sperm formation and semen quality as well spermatogenesis, ultimately reducing fertility. In females, EEDs can affect uterine development and the expression levels of reproduction-related genes, ultimately reducing female fertility and the normal development of the fetus. There are a large number of putative mechanisms by which EEDs can induce reproductive toxicity, and many studies have shown the involvement of epigenetics. In this review, we summarize the role of DNA methylation, noncoding RNAs, genomic imprinting, chromatin remodeling and histone modification in the reproductive toxicity of EEDs.

Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A.

Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A, with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.

Transgenerational Inheritance of Betaine-Induced Epigenetic Alterations in Estrogen-Responsive IGF-2/IGFBP2 Genes in Rat Hippocampus.

SCOPE: Betaine serves as a methyl donor for DNA methylation. Here, the effects of betaine on hippocampal expression of neurogenesis genes and their DNA methylation status across three generations are investigated. METHODS AND RESULTS: Pregnant rats (F0) are fed control and betaine-supplemented diets throughout gestation and lactation. Female F1 and F2 offspring at weaning, together with the F0 dams, are used in the study. Hippocampal expression of aromatase, estrogen receptor α, and estrogen-related receptor ß is downregulated in F1, together with the estrogen-responsive insulin-like growth factor 2/insulin-like growth factor binding protein 2 (IGF-2/IGFBP2) genes. However, all these genes are upregulated in F2, which follows the same pattern of F0. In agreement with changes in mRNA expression, the imprinting control region (ICR) of IGF-2 gene is hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all the affected genes is hypermethylated in F1 but hypomethylated in F2 and F0. Methyl transfer enzymes, such as betaine homocysteine methyltransferase and DNA methyltransferase 1, follow the same pattern of transgenerational inheritance. CONCLUSION: These results indicate that betaine exerts a transgenerational effect on hippocampal expression of estrogen-responsive genes in rat offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes.

[Late manifested sequelae of medications in the critical periods of development. The widening of the faulty hormonal imprinting conception]. / Az egyedfejlodés kritikus fázisaiban történo gyógyszeres kezelések késoi következményei. A hibás hormonális imprinting koncepciójának kiterjesztése.

Hormonal imprinting is a physiological process, which is a part of the receptor-hormone complex development. It determines the binding capacity of the receptors across the lifespan. It takes place perinatally in the critical period of hormone receptor development, when the developmental window for imprinting is open and permits the binding of hormone-like molecules (related or synthetic hormones, endocrine disruptors etc.) causing disturbances of the endocrine system, and the systems- influenced organs by it, for life. This is the faulty hormonal imprinting. However, studying the medical database, PubMed, a lot of data can be found on the harmful late (adult age) effects of medication in the critical period of development with non-hormonal molecules, which are manifested later in functional alterations or diseases. This could mean that in the process of faulty imprinting, the openness of the developmental window could be more important than the structural similarity of a molecule to hormones. As developmentally critical period for faulty imprinting by hormone-like molecules is not exclusively the perinatal one (this is justified in the case of faulty hormonal imprinting), the pubertal period was also studied from this aspect and similarities to the impact of perinatal use have been found (this could be called "Pubertal Origin of Health and Disease = POHaD). While in the case of hormonal faulty imprinting, the mechanism seems to be clear (considering the role of receptors), the mechanism of drug-provoked imprinting is presently uncleared (considering the variety of medications which cause late-manifested alterations). The medicaments-caused faulty imprinting conception calls attention to the dangers of medication in the perinatal as well as the pubertal periods. Orv Hetil. 2020; 161(2): 43-49.

Molecular subtype and growth hormone effects on dysmorphology in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) affects 1/15,000-1/30,000 live births and is characterized by lack of expression of paternally inherited genes on 15q11.2-15q13 caused by paternal deletions, maternal uniparental disomy (UPD), or imprinting defects. Affected individuals have distinct physical features, and growth hormone (GH) deficiency occurs in some individuals with PWS. The aim of this study is to test the hypotheses that (a) individuals with deletions and UPD have different physical and dysmorphic features, (b) individuals treated with GH have different physical and dysmorphic features than those not treated, and (c) GH treatment effects are different for individuals with UPD in comparison to those with deletions. Study participants included 30 individuals with deletions or UPD, who did or did not have GH treatment. Participants' molecular abnormalities were determined by molecular and cytogenetic analysis. Clinical data were obtained by a single dysmorphologist. Individuals with deletions were found to be heavier (p = .001), taller (p = .031), with smaller head circumferences (p = .042) and were more likely to have fair skin and hair than their family members (p = .031, .049, respectively) compared to UPD patients. Females with deletions more commonly had hypoplastic labia minora (p = .009) and clitoris (.030) in comparison to those with UPD. Individuals who received GH in both deletion and UPD groups were taller (p = .004), had larger hands (p = .011) and feet (p = .006) and a trend for a larger head circumference (p = .103). Interestingly, the GH-treated group also had a lower rate of strabismus (esotropia [p = .017] and exotropia [p = .039]). This study showed statistically significant correlations between phenotype and molecular subtypes and also between phenotype and GH treatment.

Effects of 2,3',4,4'5-pentachlorobiphenyl exposure during pregnancy on epigenetic imprinting and maturation of offspring's oocytes in mice.

Polychlorinated biphenyls (PCBs) are a class of organic pollutants that have been widely found in the environment. The chemical 2,3',4,4'5-pentachlorobiphenyl (PCB118) is an important dioxin-like PCB compound with strong toxicity. PCB118 can accumulate in adipose tissue, serum and milk in mammals, and it is highly enriched in the follicular fluid. In this study, pregnant mice were exposed to 0, 20 and 100 µg/kg/day of PCB118 during pregnancy at the fetal primordial germ cell migration stage. The methylation patterns of the imprinted genes H19, Snrpn, Peg3 and Igf2r as well as the expression levels of Dnmt1, 3a, 3b and 3l, Uhrf1, Tet2 and Tet3 in fully grown germinal vesicle oocytes were measured in offspring. The rates of in vitro maturation, in vitro fertilization, oocyte spindle and chromosomal abnormalities were also calculated. The results showed that prenatal exposure to PCB118 altered the DNA methylation status of differentially methylated regions in some imprinted genes, and the expression levels of Dnmt1, 3a, and 3l, Uhrf1 and Tet3 were also changed. In addition, PCB118 disturbed the maturation process of progeny mouse oocytes in a dose-dependent manner. Therefore, attention should be paid to the potential impacts of PCB118-contaminated dietary intake during pregnancy on the offspring's reproductive health.

Role of epigenetic regulation of Igf2 and H19 in 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD)-induced ovarian toxicity in offspring rats.

2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure during embryonic gonadal sex determination had been demonstrated to harm the ovarian development. However, its mechanism was unclear and possibly related to epigenetic regulation. In the present study, the pregnant rats were treated with TCDD (100 ng/kg/day or 500 ng/kg/day) or only vehicle and corn oil on the day 8-14 of gestation through the gavage with a stainless-steel feeding needle. The vaginal opening time and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle count, real-time PCR, Western Blotting and DNA methylation analysis for Igf2 and H19 were carried out. The results showed that maternal TCDD exposure disrupted estrous cyclicity, resulted in aberrant concentration of serum E2 and FSH, and affected the number of primordial follicles, secondary follicles and corpus luteum. However, TCDD had no effect on the number of primary follicles and atresia follicles. Furthermore, the mRAN expression of imprinted genes Igf2 and H19 was down-regulated, and the IGF2 protein was also down-regulated. TCDD exposure did not alter the mean methylation rate of Igf2 DMR2 and H19 ICR, and only some CpG sites throughout them were hypermethylated in high-dose TCDD rats. In conclusion, maternal exposure of TCDD could affect the ovary development and functions which were possibly associated with down-regulation expression of IGF2 and H19. However, it was not entirely clear whether the impairment of ovary by TCDD was related to the methylation pattern of Igf2 and H19 ICR.

Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model.

STUDY QUESTION: Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)? SUMMARY ANSWER: Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes. WHAT IS KNOWN ALREADY: Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear. STUDY DESIGN, SIZE, DURATION: Outbred female mice were fed three folic acid-supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilisation, embryo culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1 and H19) in matched midgestation embryos and placentas (n = 31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in placentas (n = 6 normal placentas per sex/group) and embryos (n = 6 normal female embryos/group; n = 3 delayed female embryos/group) using reduced representation bisulfite sequencing (RRBS). MAIN RESULTS AND THE ROLE OF CHANCE: Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were more pronounced in males. LARGE-SCALE DATA: The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143. LIMITATIONS REASONS FOR CAUTION: Although the combination of mouse ART utilised in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account foetal sex, are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our findings support moderation in the dose of folic acid supplements taken during ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest.

Human-induced pluripotent stems cells as a model to dissect the selective neurotoxicity of methylmercury.

Methylmercury (MeHg) is a potent neurotoxicant affecting both the developing and mature central nervous system (CNS) with apparent indiscriminate disruption of multiple homeostatic pathways. However, genetic and environmental modifiers contribute significant variability to neurotoxicity associated with human exposures. MeHg displays developmental stage and neural lineage selective neurotoxicity. To identify mechanistic-based neuroprotective strategies to mitigate human MeHg exposure risk, it will be critical to improve our understanding of the basis of MeHg neurotoxicity and of this selective neurotoxicity. Here, we propose that human-based pluripotent stem cell cellular approaches may enable mechanistic insight into genetic pathways that modify sensitivity of specific neural lineages to MeHg-induced neurotoxicity. Such studies are crucial for the development of novel disease modifying strategies impinging on MeHg exposure vulnerability.

The genomic imprint of cancer therapies helps timing the formation of metastases.

A retrospective determination of the time of metastasis formation is essential for a better understanding of the evolution of oligometastatic cancer. This study was based on the hypothesis that genomic alterations induced by cancer therapies could be used to determine the temporal order of the treatment and the formation of metastases. We analysed the whole genome sequence of a primary tumour sample and three metastatic sites derived from autopsy samples from a young never-smoker lung adenocarcinoma patient with an activating EGFR mutation. Mutation detection methods were refined to accurately detect and distinguish clonal and subclonal mutations. In comparison to a panel of samples from untreated smoker or never-smoker patients, we showed that the mutagenic effect of cisplatin treatment could be specifically detected from the base substitution mutations. Metastases that arose before or after chemotherapeutic treatment could be distinguished based on the allele frequency of cisplatin-induced dinucleotide mutations. In addition, genomic rearrangements and late amplification of the EGFR gene likely induced by afatinib treatment following the acquisition of a T790M gefitinib resistance mutation provided further evidence to tie the time of metastasis formation to treatment history. The established analysis pipeline for the detection of treatment-derived mutations allows the drawing of tumour evolutionary paths based on genomic data, showing that metastases may be seeded well before they become detectable by clinical imaging.

Bone Manifestation of Faulty Perinatal Hormonal Imprinting: A Review.

Hormonal imprinting takes place at the first encounter between the developing receptor and its target hormone and the encounter determines the receptor's binding capacity for life. In the critical period of development, when the window for imprinting is open, the receptor can be misdirected by related hormones, synthetic hormones, and industrial or communal endocrine disruptors which cause faulty hormonal imprinting with life-long consequences. Considering these facts, the hormonal imprinting is a functional teratogen provoking alterations in the perinatal (early postnatal) period. One single encounter with a low dose of the imprinter in the critical developmental period is enough for the formation of faulty imprinting, which is manifested later, in adult age. This has been justified in the immune system, in sexuality, in animal behavior and brain neurotransmitters etc. by animal experiments and human observations. This review points to the faulty hormonal imprinting in the case of bones (skeleton), by single or repeated treatments. The imprinting is an epigenetic alteration which is inherited to the progeny generations. From clinical aspect, the faulty imprinting can have a role in the pathological development of the bones as well, as in the risk of osteoporotic fractures, etc.

Environmental effects on chromatin repression at imprinted genes and endogenous retroviruses.

Environmental factors can perturb epigenetic regulation. In mammals, most studies have focused on repressive DNA methylation. Two attractive model systems to monitor environmentally triggered drifts in DNA methylation are genomic imprinting and endogenous retroviruses (ERVs), particularly intracisternal-A particles (IAPs). These systems show mechanistic similarities in their repressive chromatin organization, which in somatic cells is comparable between the DNA-methylated alleles of imprinted differentially methylated regions (DMRs) and repressed ERVs. Here, we present how during development, nutrition and chemical components can perturb DNA methylation at imprinted genes and ERVs, and discuss the still poorly understood underlying mechanisms.

Early prenatal alcohol exposure alters imprinted gene expression in placenta and embryo in a mouse model.

Prenatal alcohol exposure (PAE) can harm the embryonic development and cause life-long consequences in offspring's health. To clarify the molecular mechanisms of PAE we have used a mouse model of early alcohol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first eight days of gestation (GD 0.5-8.5). Owing to the detected postnatal growth-restricted phenotype in the offspring of this mouse model and both prenatal and postnatal growth restriction in alcohol-exposed humans, we focused on imprinted genes Insulin-like growth factor 2 (Igf2), H19, Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn) and Paternally expressed gene 3 (Peg3), which all are known to be involved in embryonic and placental growth and development. We studied the effects of alcohol on DNA methylation level at the Igf2/H19 imprinting control region (ICR), Igf2 differentially methylated region 1, Snrpn ICR and Peg3 ICR in 9.5 embryonic days old (E9.5) embryos and placentas by using MassARRAY EpiTYPER. To determine alcohol-induced alterations globally, we also examined methylation in long interspersed nuclear elements (Line-1) in E9.5 placentas. We did not observe any significant alcohol-induced changes in DNA methylation levels. We explored effects of PAE on gene expression of E9.5 embryos as well as E9.5 and E16.5 placentas by using quantitative PCR. The expression of growth promoter gene Igf2 was decreased in the alcohol-exposed E9.5 and E16.5 placentas. The expression of negative growth controller H19 was significantly increased in the alcohol-exposed E9.5 embryos compared to controls, and conversely, a trend of decreased expression in alcohol-exposed E9.5 and E16.5 placentas were observed. Furthermore, increased Snrpn expression in alcohol-exposed E9.5 embryos was also detected. Our study indicates that albeit no alterations in the DNA methylation levels of studied sequences were detected by EpiTYPER, early PAE can affect the expression of imprinted genes in both developing embryo and placenta.

Effects of Cadmium Exposure on DNA Methylation at Imprinting Control Regions and Genome-Wide in Mothers and Newborn Children.

BACKGROUND: Imprinted genes are defined by their preferential expression from one of the two parental alleles. This unique mode of gene expression is dependent on allele-specific DNA methylation profiles established at regulatory sequences called imprinting control regions (ICRs). These loci have been used as biosensors to study how environmental exposures affect methylation and transcription. However, a critical unanswered question is whether they are more, less, or equally sensitive to environmental stressors as the rest of the genome. OBJECTIVES: Using cadmium exposure in humans as a model, we aimed to determine the relative sensitivity of ICRs to perturbation of methylation compared to similar, nonimprinted loci in the genome. METHODS: We assayed DNA methylation genome-wide using bisulfite sequencing of 19 newborn cord blood and 20 maternal blood samples selected on the basis of maternal blood cadmium levels. Differentially methylated regions (DMRs) associated with cadmium exposure were identified. RESULTS: In newborn cord blood and maternal blood, 641 and 1,945 cadmium-associated DMRs were identified, respectively. DMRs were more common at the 15 maternally methylated ICRs than at similar nonimprinted loci in newborn cord blood (p=5.64×10-8) and maternal blood (p=6.22×10-14), suggesting a higher sensitivity for ICRs to cadmium. Genome-wide, Enrichr analysis indicated that the top three functional categories for genes that overlapped DMRs in maternal blood were body mass index (BMI) (p=2.0×10-5), blood pressure (p=3.8×10-5), and body weight (p=0.0014). In newborn cord blood, the top three functional categories were BMI, atrial fibrillation, and hypertension, although associations were not significant after correction for multiple testing (p=0.098). These findings suggest that epigenetic changes may contribute to the etiology of cadmium-associated diseases. CONCLUSIONS: We analyzed cord blood and maternal blood DNA methylation profiles genome-wide at nucleotide resolution in individuals selected for high and low blood cadmium levels in the first trimester. Our findings suggest that ICRs may be hot spots for perturbation by cadmium, motivating further study of these loci to investigate potential mechanisms of cadmium action. https://doi.org/10.1289/EHP2085.

Maternal levels of endocrine disrupting chemicals in the first trimester of pregnancy are associated with infant cord blood DNA methylation.

Endocrine disrupting chemicals (EDCs) pose a public health risk through disruption of normal biological processes. Identifying toxicoepigenetic mechanisms of developmental exposure-induced effects for EDCs, such as phthalates or bisphenol A (BPA), is essential. Here, we investigate whether maternal exposure to EDCs is predictive of infant DNA methylation at candidate gene regions. In the Michigan Mother-Infant Pairs (MMIP) cohort, DNA was extracted from cord blood leukocytes for methylation analysis by pyrosequencing (n = 116) and methylation changes related to first trimester levels of 9 phthalate metabolites and BPA. Growth and metabolism-related genes selected for methylation analysis included imprinted (IGF2, H19) and non-imprinted (PPARA, ESR1) genes along with LINE-1 repetitive elements. Findings revealed decreases in methylation of LINE-1, IGF2, and PPARA with increasing phthalate concentrations. For example, a log unit increase in ΣDEHP corresponded to a 1.03 [95% confidence interval (CI): -1.83, -0.22] percentage point decrease in PPARA methylation. Changes in DNA methylation were also inversely correlated with PPARA gene expression determined by RT-qPCR (r = -0.34, P = 0.02), thereby providing evidence in support of functional relevance. A sex-stratified analysis of EDCs and DNA methylation showed that some relationships were female-specific. For example, urinary BPA exposure was associated with a 1.35 (95%CI: -2.69, -0.01) percentage point decrease in IGF2 methylation and a 1.22 (95%CI: -2.27, -0.16) percentage point decrease in PPARA methylation in females only. These findings add to a body of evidence suggesting epigenetically labile regions may provide a conduit linking early exposures with disease risk later in life and that toxicoepigenetic susceptibility may be sex specific.

Mitotically Stable Modification of DNA Methylation in IGF2/H19 Imprinting Control Region Is Associated with Activated Hepatic IGF2 Expression in Offspring Rats from Betaine-Supplemented Dams.

The growth-promoting action of betaine involves activation of GH/IGF-1 signaling, yet it remains unclear whether insulin-like growth factor 2 (IGF2), an imprinting gene, is affected by maternal dietary betaine supplementation. In this study, F1 offspring rats derived from dams fed basal or betaine-supplemented diet were examined at D21 and D63. Maternal betaine significantly upregulated the hepatic expression of IGF2 mRNA and protein in offspring rats at both D21 and D63, which was accompanied by enhanced hepatic IGF2 immunoreactivity and elevated serum IGF-2 level. Higher protein expression of betaine-homocysteine methyltransferase and DNA methyltransferase 1 was detected in the betaine group at D21, but not D63. However, hypermethylation of the imprinting control region of the IGF2/H19 locus at D21 was maintained at D63. These results indicate that maternal betaine modifies DNA methylation of IGF2/H19 imprinting control region in a mitotically stable fasion, which was associated with the activation hepatic IGF2 expression in offspring rats.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels.

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis -including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting (GRB10, GNAS, XIST) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified.

Curcumin Suppresses In Vitro Proliferation and Invasion of Human Prostate Cancer Stem Cells by Modulating DLK1-DIO3 Imprinted Gene Cluster MicroRNAs.

AIMS: Curcumin can suppress human prostate cancer (HuPCa) cell proliferation and invasion. However, it is not known whether curcumin can inhibit HuPCa stem cell (HuPCaSC) proliferation and invasion. MATERIALS AND METHODS: We used methyl thiazolyl tetrazolium and Transwell assays to examine the proliferation and invasion of the HuPCaSC lines DU145 and 22Rv1 following curcumin or dimethyl sulfoxide (control) treatment. The microRNA (miRNA) expression levels in the DLK1-DIO3 imprinted genomic region in the cells and in tumor tissues from patients with PCa were examined using microarray and quantitative PCR. RESULTS: The median inhibitory concentration of curcumin for HuPCa cells significantly inhibited HuPCaSC proliferation and invasion in vitro. The miR-770-5p and miR-1247 expression levels in the DLK1-DIO3 imprinted gene cluster were significantly different between the curcumin-treated and control HuPCaSCs. Overexpression of these positive miRNAs significantly increased the inhibition rates of miR-770-5p- and miR-1247-transfected HuPCaSCs compared to the control miR-Mut-transfected HuPCaSCs. Lastly, low-tumor grade PCa tissues had higher miR-770-5p and miR-1247 expression levels than high-grade tumor tissues. CONCLUSIONS: Curcumin can suppress HuPCaSC proliferation and invasion in vitro by modulating specific miRNAs in the DLK1-DIO3 imprinted gene cluster.

Dysregulation of DNA methylation and expression of imprinted genes in mouse placentas of fetal growth restriction induced by maternal cadmium exposure.

Cadmium (Cd) is one of the most toxic environmental pollutants that cause fetal malformation and growth restriction. However, the molecular mechanisms underlying maternal Cd toxicity on fetal growth remain largely unknown. Specifically, the expression profiles and the regulation mechanisms of the imprinted genes, have been poorly characterized in the etiology of Cd-induced fetal growth restriction (FGR). In the present study, 13 imprinted genes associated with the fetal growth and placenta development were selected and their expression patterns were examined in the Cd-exposed placentas. Quantitative real-time PCR and western blot results showed that the maternally expressed gene, Cyclin dependent kinase inhibitor 1c (Cdkn1c), and paternally expressed gene, Paternally expressed gene 10 (Peg10), were significantly upregulated and downregulated respectively in the Cd-exposed placentas when compared to the normal ones respectively. Moreover, data from bisulfate PCR demonstrated the changes of the methylation levels of the promoter regions of Cdkn1c and Peg10 in the Cd-exposed placentas. In addition, the expression profile of Cdkn1c was correlated with the methylation levels of site 2 (-837--692) but not site 1 (-389--185) of its promoter region. Therefore, our results suggest that changes of the DNA methylation levels of the promoter regions and the expression patterns of Cdkn1c and Peg10 may be involved in the etiology of Cd-induced fetal growth restriction.