Search

Background A critical question emerging in the field of developmental toxicology is whether alterations in chromatin structure induced by toxicant exposure control patterns of gene expression or, instead, are structural changes that are part of a nuclear stress response. Previously, we used a mouse model to conduct a three-way comparison between control offspring, alcohol-exposed but phenotypically normal animals, and alcohol-exposed offspring exhibiting craniofacial and central nervous system structural defects. In the cerebral cortex of animals exhibiting alcohol-induced dysgenesis, we identified a dramatic increase in the enrichment of dimethylated histone H3, lysine 9 (H3K9me2) within the regulatory regions of key developmental factors driving histogenesis in the brain. However, whether this change in chromatin structure is causally involved in the development of structural defects remains unknown. Results Deep-sequencing analysis of the cortex transcriptome reveals that the emergence of alcohol-induced structural defects correlates with disruptions in the genetic pathways controlling oxidative phosphorylation and mitochondrial function. The majority of the affected pathways are downstream targets of the mammalian target of rapamycin complex 2 (mTORC2), indicating that this stress-responsive complex plays a role in propagating the epigenetic memory of alcohol exposure through gestation. Importantly, transcriptional disruptions of the pathways regulating oxidative homeostasis correlate with the emergence of increased H3K9me2 across genic, repetitive, and non-transcribed regions of the genome. However, although associated with gene silencing, none of the candidate genes displaying increased H3K9me2 become transcriptionally repressed, nor do they exhibit increased markers of canonical heterochromatin. Similar to studies in C. elegans, disruptions in oxidative homeostasis induce the chromatin looping factor SATB2, but in mammals, this protein does not appear to drive increased H3K9me2 or altered patterns of gene expression. Conclusions Our studies demonstrate that changes in H3K9me2 associate with alcohol-induced congenital defects, but that this epigenetic change does not correlate with transcriptional suppression. We speculate that the mobilization of SATB2 and increased enrichment of H3K9me2 may be components of a nuclear stress response that preserve chromatin integrity and interactions under prolonged oxidative stress. Further, we postulate that while this response may stabilize chromatin structure, it compromises the nuclear plasticity required for normal differentiation.

Objectives Paternally inherited alterations in epigenetic programming are emerging as relevant factors in numerous disease states, including the growth and metabolic defects observed in fetal alcohol spectrum disorders. In rodents, chronic paternal alcohol use induces fetal growth restriction, as well as sex-specific alterations in insulin signaling and lipid homeostasis in the offspring. Based on previous studies, we hypothesized that the observed metabolic irregularities are the consequence of paternally inherited alterations liver x receptor (LXR) activity. Methods Male offspring of alcohol-exposed sires were challenged with a high-fat diet and the molecular pathways controlling glucose and lipid homeostasis assayed for LXR-induced alterations. Results Similar to findings in studies employing LXR agonists we found that the male offspring of alcohol-exposed sires display resistance to diet-induced obesity and improved glucose homeostasis when challenged with a high-fat diet. This improved metabolic adaptation is mediated by LXRα trans-repression of inflammatory cytokines, releasing IKKβ inhibition of the insulin signaling pathway. Interestingly, paternally programmed increases in LXRα expression are liver-specific and do not manifest in the pancreas or visceral fat. Conclusions These studies identify LXRα as a key mediator of the long-term metabolic alterations induced by preconception paternal alcohol use., Highlights • Chronic paternal alcohol use induces up-regulation of LXRα in the male offspring. • Male offspring of alcohol-exposed fathers are protected from diet-induced obesity. • Paternally-inherited up-regulation of LXRα only manifests in the liver. • Improved metabolic adaptation is linked to LXRα suppression of cytokine production. • Male offspring exhibit the same phenotypes observed in studies of LXR agonists.

Epigenetic mechanisms of paternal inheritance are an emerging area of interest in our efforts to understand fetal alcohol spectrum disorders. In rodent models examining maternal alcohol exposures, different maternal genetic backgrounds protect or sensitize offspring to alcohol-induced teratogenesis. However, whether maternal background can mitigate sperm-inherited alterations in developmental programming and modify the penetrance of growth defects induced by preconception paternal alcohol exposures remains unaddressed. In our previous studies examining pure C57Bl/6J crosses, the offspring of alcohol-exposed sires exhibited fetal growth restriction, enlarged placentas, and decreased placental efficiency. Here, we find that in contrast to our previous studies, the F1 offspring of alcohol-exposed C57Bl/6J sires and CD-1 dams do not exhibit fetal growth restriction, with male fetuses developing smaller placentas and increased placental efficiencies. However, in these hybrid offspring, preconception paternal alcohol exposure induces sex-specific changes in placental morphology. Specifically, the female offspring of alcohol-exposed sires displayed structural changes in the junctional and labyrinth zones, along with increased placental glycogen content. These changes in placental organization are accompanied by female-specific alterations in the expression of imprinted genes Cdkn1c and H19. Although male placentae do not display overt changes in placental histology, using RNA-sequencing, we identified programmed alterations in genes regulating oxidative phosphorylation, mitochondrial function, and Sirtuin signaling. Collectively, our data reveal that preconception paternal alcohol exposure transmits a stressor to developing offspring, that males and females exhibit distinct patterns of placental adaptation, and that maternal genetic background can modulate the effects of paternal alcohol exposure.