Variably methylated retrotransposons are refractory to a range of environmental perturbations.

The agouti viable yellow (Avy) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. Avy expressivity is sensitive to a range of early-life chemical exposures and nutritional interventions, suggesting that environmental perturbations can have long-lasting effects on the methylome. However, the extent to which VM-IAP elements are environmentally labile with phenotypic implications is unknown. Using a recently identified repertoire of VM-IAPs, we assessed the epigenetic effects of different environmental contexts. A longitudinal aging analysis indicated that VM-IAPs are stable across the murine lifespan, with only small increases in DNA methylation detected for a subset of loci. No significant effects were observed after maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation. A genetic mouse model of abnormal folate metabolism exhibited shifted VM-IAP methylation levels and altered VM-IAP-associated gene expression, yet these effects are likely largely driven by differential targeting by polymorphic KRAB zinc finger proteins. We conclude that epigenetic variability at retrotransposons is not predictive of environmental susceptibility.

Mice exposed to bisphenol A exhibit depressive-like behavior with neurotransmitter and neuroactive steroid dysfunction.

Fetal exposure to endocrine disrupting chemicals (EDCs) has been associated with adverse neurobehavioral outcomes across the lifespan and can persist across multiple generations of offspring. However, the underlying mechanisms driving these changes are not well understood. We investigated the molecular perturbations associated with EDC-induced behavioral changes in first (F1) and second (F2) filial generations, using the model EDC bisphenol A (BPA). C57BL/6J dams were exposed to BPA from preconception until lactation through the diet at doses (10 µg/kg bw/d-lower dose or 10 mg/kg bw/d-upper dose) representative of human exposure levels. As adults, F1 male offspring exhibited increased depressive-like behavior, measured by the forced swim test, while females were unaffected. These behavioral changes were limited to the F1 generation and were not associated with altered maternal care. Transcriptome analysis by RNA-sequencing in F1 control and upper dose BPA-exposed adult male hippocampus revealed neurotransmitter systems as major pathways disrupted by developmental BPA exposure. High performance liquid chromatography demonstrated a male-specific reduction in hippocampal serotonin. Administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg bw) rescued the depressive-like phenotype in males exposed to lower, but not upper, dose BPA, suggesting distinct mechanisms of action for each exposure dose. Finally, high resolution mass spectrometry revealed reduced circulating levels of the neuroactive steroid dehydroepiandrosterone in BPA-exposed males, suggesting another potential mechanism underlying the depressive-like phenotype. Thus, behavioral changes associated with early life BPA exposure may be mediated by sex-specific disruptions in the serotonergic system and/or sex steroid biogenesis in male offspring.

Bile Acids and Tryptophan Metabolism Are Novel Pathways Involved in Metabolic Abnormalities in BPA-Exposed Pregnant Mice and Male Offspring.

Increasing evidence has demonstrated that exposure to endocrine-disrupting chemicals impacts maternal and fetal health, but the underlying mechanisms are still unclear. We previously showed that dietary exposure to 10 µg/kg body weight (bw)/d and 10 mg/kg bw/d of bisphenol A (BPA) during pregnancy induced metabolic abnormalities in F1 male offspring and gestational glucose intolerance in F0 pregnant mice. The aim of this study was to elucidate the underlying etiologies of BPA exposure-induced metabolic disease by analyzing the male fetal liver metabolome. Using the Metabolon Discover HD4 Platform, our laboratory identified metabolic pathways that were altered by BPA exposure, including biochemicals in lipid and amino acid metabolism. Specifically, primary and secondary bile acids were increased in liver from BPA-exposed embryonic day 18.5 male fetuses. We subsequently showed that increased bile acid was associated with a defective farnesoid X receptor-dependent negative feedback mechanism in BPA-exposed fetuses. In addition, through metabolomics, we observed that BPA-exposed fetuses had elevated tryptophan levels. Independent liquid chromatography and mass spectrometry measurement revealed that BPA-exposed dams also had increased tryptophan levels relative to those of controls. Because several key enzymes in tryptophan catabolism are vitamin B6 dependent and vitamin B6 deficiencies have been linked to gestational diabetes, we tested the impact of vitamin B6 supplementation and showed that it rescued gestational glucose intolerance in BPA-exposed pregnant mice. Our study has therefore identified two pathways (bile acid and tryptophan metabolism) that potentially underlie BPA-induced maternal and fetal metabolic disease.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos.

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.