Paternal alcohol exposures program intergenerational hormetic effects on offspring fetoplacental growth.

Hormesis refers to graded adaptive responses to harmful environmental stimuli where low-level toxicant exposures stimulate tissue growth and responsiveness while, in contrast, higher-level exposures induce toxicity. Although the intergenerational inheritance of programmed hormetic growth responses is described in plants and insects, researchers have yet to observe this phenomenon in mammals. Using a physiologically relevant mouse model, we demonstrate that chronic preconception paternal alcohol exposures program nonlinear, dose-dependent changes in offspring fetoplacental growth. Our studies identify an inverse j-shaped curve with a threshold of 2.4 g/Kg per day; below this threshold, paternal ethanol exposures induce programmed increases in placental growth, while doses exceeding this point yield comparative decreases in placental growth. In male offspring, higher paternal exposures induce dose-dependent increases in the placental labyrinth layer but do not impact fetal growth. In contrast, the placental hypertrophy induced by low-level paternal ethanol exposures associate with increased offspring crown-rump length, particularly in male offspring. Finally, alterations in placental physiology correlate with disruptions in both mitochondrial-encoded and imprinted gene expression. Understanding the influence of ethanol on the paternally-inherited epigenetic program and downstream hormetic responses in offspring growth may help explain the enormous variation observed in fetal alcohol spectrum disorder (FASD) phenotypes and incidence.

Alcohol induced increases in sperm Histone H3 lysine 4 trimethylation correlate with increased placental CTCF occupancy and altered developmental programming.

Using a mouse model, studies by our group reveal that paternal preconception alcohol intake affects offspring fetal-placental growth, with long-lasting consequences on adult metabolism. Here, we tested the hypothesis that chronic preconception male alcohol exposure impacts histone enrichment in sperm and that these changes are associated with altered developmental programming in the placenta. Using chromatin immunoprecipitation, we find alcohol-induced increases in sperm histone H3 lysine 4 trimethylation (H3K4me3) that map to promoters and presumptive enhancer regions enriched in genes driving neurogenesis and craniofacial development. Given the colocalization of H3K4me3 with the chromatin binding factor CTCF across both sperm and embryos, we next examined CTCF localization in the placenta. We find global changes in CTCF binding within placentae derived from the male offspring of alcohol-exposed sires. Furthermore, altered CTCF localization correlates with dysregulated gene expression across multiple gene clusters; however, these transcriptional changes only occur in male offspring. Finally, we identified a correlation between genomic regions exhibiting alcohol-induced increases in sperm H3K4me3 and increased CTCF binding in male placentae. Collectively, our analysis demonstrates that the chromatin landscape of sperm is sensitive to chronic alcohol exposure and that a subset of these affected regions exhibits increased placental CTCF enrichment.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome.

BACKGROUND: Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. RESULTS: Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. CONCLUSIONS: These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Maternal background alters the penetrance of growth phenotypes and sex-specific placental adaptation of offspring sired by alcohol-exposed males.

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.

Programmed increases in LXRα induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity.

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 IKKß 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.

DNA methylation-independent growth restriction and altered developmental programming in a mouse model of preconception male alcohol exposure.

The preconception environment is a significant modifier of dysgenesis and the development of environmentally-induced disease. To date, fetal alcohol spectrum disorders (FASDs) have been exclusively associated with maternal exposures, yet emerging evidence suggests male-inherited alterations in the developmental program of sperm may be relevant to the growth-restriction phenotypes of this condition. Using a mouse model of voluntary consumption, we find chronic preconception male ethanol exposure associates with fetal growth restriction, decreased placental efficiency, abnormalities in cholesterol trafficking, sex-specific alterations in the genetic pathways regulating hepatic fibrosis, and disruptions in the regulation of imprinted genes. Alterations in the DNA methylation profiles of imprinted loci have been identified in clinical studies of alcoholic sperm, suggesting the legacy of paternal drinking may transmit via heritable disruptions in the regulation of imprinted genes. However, the capacity of sperm-inherited changes in DNA methylation to broadly transmit environmentally-induced phenotypes remains unconfirmed. Using bisulphite mutagenesis and second-generation deep sequencing, we find no evidence to suggest that these phenotypes or any of the associated transcriptional changes are linked to alterations in the sperm-inherited DNA methylation profile. These observations are consistent with recent studies examining the male transmission of diet-induced phenotypes and emphasize the importance of epigenetic mechanisms of paternal inheritance beyond DNA methylation. This study challenges the singular importance of maternal alcohol exposures and suggests paternal alcohol abuse is a significant, yet overlooked epidemiological factor complicit in the genesis of alcohol-induced growth defects, and may provide mechanistic insight into the failure of FASD children to thrive postnatally.

Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure.

Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact that alcohol and other drugs of abuse exert consistently notes a disconnection between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the 'histone code' induced by prenatal exposures to alcohol implicitly subvert gene expression, or whether the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation, or affect the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest that changes in chromatin structure cannot unequivocally explain dysgenesis in isolation.