Session 3 Abstracts
OPENING TALK
Evidence for Germline Non-genetic Inheritance of Human Phenotypes and Diseases
• Matthew Smith-Raska, MD, PhD, Weill Cornell Medical, NY, USA
Evidence for DNA sequence-independent inheritance of phenotypes and diseases continues to grow in model organisms, in which a well-defined exposure leads to epigenetic abnormalities in germ cells and ultimately phenotypic defects in descendants. In humans, there are fewer reports of this phenomenon, due to a variety of complications in evaluating this mechanism of inheritance in humans, both in the examination of germ cells after an exposure as well as the detection of phenotypes in descendants. Discovery of these processes in humans remains challenging because human populations are heterogenous, most phenotypes are the consequence of many additive influences, and the correlation of a phenotype in an adult with a specific exposure in their descendants requires excellent record-keeping over multiple decades. Despite these limitations, this phenomenon is emerging as a possible explanation for the many diseases that have evidence of strong heritability that cannot be explained by DNA variants, and there is mounting evidence that non-DNA factors inherited from germ cells are critical contributors to numerous human disease processes.
SHORT TALKS
Chairs: Mathia Colwell and Patrick Allard
Paternal Drug Exposure Reveals Previously Unconsidered Class of Sperm RNA as Novel Candidate in RNA-mediated Inheritance
• Katharina Gapp, PhD, Center for Neuroscience Zürich ETHZ and Uni Zürich
Trauma is a risk factor for the development of neuropsychiatric disease later in life. Rodent studies suggest that such risk can extend to the offspring not directly exposed. Non-genetic disease risk inheritance of chronic exposures often involves alterations and transmission of sperm RNA. While acute exaggerated stress is known to also affect behavioral abnormalities its effect on germline and offspring behavior is unknown. Here we investigate the effects of acute activation of stress-sensitive Glucocorticoid receptor using Dexamethasone a drug of common clinical use on the germline and offspring metabolism. We find that acute Dexamethasone treatment, affects the RNA payload of postmeiotic mature sperm as soon as 3 hours post exposure, challenging the prevalent view that epididymal transit is required to alter sperm RNA. The drug further changes early embryonic transcriptional trajectories of the offspring, as determined by single embryo sequencing, and impacts metabolism in the offspring. Our data reveal persistent regulation of tRNA fragments in sperm and the descendant 2-cell- embryos, suggesting actual transmission from sperm to embryo. Lastly, we describe environmentally induced alterations in the previously unconsidered class of sperm RNAs, and their targets in the early embryo, highlighting this class as a novel candidate in RNA-mediated inheritance.
Transgenerational Transmission of Behavioral Phenotypes in a Mouse Model of Paternal Saccharin and Nicotine Exposure
• Dierdre McCarthy, Florida State University, Tallahassee, FL
Non-nutritive sweeteners such as saccharin are regarded as safe for human consumption and their use is widely prevalent. Although saccharin is not metabolized in the body it activates brain’s reward circuitry suggesting that it may influence neurobehavioral phenotypes. Using a paternal saccharin exposure mouse model, we found that saccharin exposure produced motor-impulsivity in the saccharin-exposed male mice as well as in their offspring. In addition, the offspring showed locomotor hyperactivity which was not observed in fathers. The stimulant drug methylphenidate mitigated the hyperactivity, highlighting dopamine’s role in this behavior. Heritability was influenced by two factors: Sex and the duration of paternal exposure. F1 male but not female offspring derived from 12- but not 8-weeks of paternal exposure produced heritable phenotypes. Spermatazoal DNA was hypermethylated in the saccharin-exposed fathers, especially at dopamine receptor promoter regions, suggesting that epigenetic modification of germ cell DNA may mediate transgenerational transmission of behavioral phenotypes. Nicotine is another substance that is widely used. Nicotine use via traditional cigarettes, smokeless tobacco products, and electronic cigarettes is on the rise, especially among the youth and young adults. Some nicotine products contain saccharin exposing the user to saccharin and nicotine. We found that co-exposure of male mice to saccharin and nicotine also produced significant behavioral impairment in their offspring. Thus, exposure to saccharin alone or saccharin and nicotine has potential adverse neurobehavioral consequences for the exposed individuals and their descendants.
The National Birth Defect Registry, a Novel Approach to Identify Birth Defect Clusters for Case Control Studies
• Betty Mekdeci, National Birth Defect Registry
Each year, 150,000 children are born in the United States with major structural birth defects and one in seven children is diagnosed with a neurodevelopmental disorder. The United States no longer has a national birth defect surveillance system and less than half the nation's population is covered in state birth defect registries. To fill this void, in 1990 Birth Defect Research for Children created the National Birth Defect Registry, a project based on the concept of the "alert practitioner" i.e. a doctor or researcher has seen a similar pattern of birth defects in a number of children and has found a common exposure in the prenatal or pre-conceptual histories of these cases. The devastating effects of thalidomide, the rubella virus, Dilantin, DES, alcohol, mercury and other teratogens were first discovered through this method. Seven nationally prominent scientists peer-reviewed and endorsed the design and utility of the registry. Exposure, work, health and genetic histories are collected for both mothers and fathers as well as data on structural as well as functional birth defects. A section for grandchildren has also been added for collection of data from Vietnam veterans because of concern about epigenetic changes to sperm from dioxin exposure that may affect the germline. Registry data on the children of Vietnam and Gulf War veterans has been instrumental in the passage of a law in 2016 that requires government research on the effects of military toxins in the children and grandchildren of all veterans.
Developmental Cannabidiol Exposure Increases Anxiety and Modifies Genome-wide Brain DNA Methylation in Adult Female Mice
• Nicole Wanner, DVM, University of Minnesota
Use of cannabidiol (CBD), the primary non-psychoactive compound in cannabis, is rising while little is known about the consequences of developmental exposure. Previous work indicates that direct CBD exposure has anxiolytic, antidepressant, and other neuropsychiatric effects. Epigenetic perturbation is a critically important mechanism in the context of exposures during embryogenesis, when exogenous compounds can disrupt DNA methylation and impart phenotypic effects persisting into adulthood. We hypothesized that developmental CBD exposure would detrimentally impact behavior mediated in part by the epigenome. Female wild-type Agouti viable yellow (Avy) mice were exposed to 20 mg/kg CBD or vehicle daily from two weeks prior to mating through lactation. Coat color shifts, a readout of DNA methylation in this strain, were measured in F1 Avy/a offspring. Young adult F1 a/a offspring were subjected to tests of working spatial memory and anxiety. Reduced-representation bisulfite sequencing was performed on F1 cerebral cortex and hippocampus to identify genome-wide changes in DNA methylation. F1 offspring exposed to CBD during development exhibited increased anxiety and improved memory behavior in a sex-specific manner. Further, thousands of differentially methylated loci (DMLs) were identified in both brain regions with functional enrichment for neurogenesis, substance use phenotypes, and other psychologically relevant terms. These findings demonstrate for the first time that despite positive effects for direct exposure, developmental CBD is associated with mixed behavioral outcomes and perturbation of the brain epigenome.
Developmental TCDD Exposure Results in Multigenerational Histological, Transcriptomic, and Methylomic Abnormalities in Male Zebrafish Gonads
• Danielle Meyer, Wayne State University
The industrial by-product TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a potent environmental toxicant and model endocrine-disrupting chemical (EDC) with known teratogenic effects on humans, rodents and fish. Developmental exposure to some EDCs, including TCDD, is linked to the occurrence of adult-onset and multigenerational disease. Our lab uses zebrafish (Danio rerio) as a model to study these effects due to their short generation time, transparency in early development, and ease of early-life exposure. Previous work in this lab revealed decreased fertility in adult zebrafish exposed to TCDD as juveniles. Because the unexposed descendants of these fish (F1 and F2) presented with reduced fertility mediated through the males, we examined the testes of TCDD-exposed males from all three generations, looking for changes in histopathology, gene expression, and whole genome methylation that could account for these reproductive deficits. Histological analysis indicated a shift in germ cell ratio towards immature spermatogonia in F0 and F1 generations, indicating delayed spermiation in exposed males and descendants. Microarray analysis of exposed testes indicated that genes involved in epigenetic modification as well as multiple reproduction and infertility pathways were affected across all generations, including testis development and spermatogenesis, lipid metabolism and steroidogenesis, oxidative stress response, citric acid cycle, peroxisome, and xenobiotic response. Upon whole genome methylation analysis, we found TCDD-induced methylation changes in reproductive genes as well as histone-modifying enzymes across multiple generations. Overall, we found that the interplay between these transcriptomic and epigenetic changes could account for the transgenerational impact on fertility due to developmental toxicant exposure.
Caenorhabditis elegans SET1/COMPASS Maintains Germline Identity by Preventing Transcriptional Deregulation Across Generations
• Valerie Robert, LBMC, ENS Lyon
Chromatin regulators contribute to the maintenance of the germline transcriptional program. In the absence of SET-2, the C. elegans homologue of the SET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational loss of germline identity, leading to sterility. To identify transcriptional signatures associated with progressive loss of fertility, we performed expression profiling of set-2 mutant germlines across generations. We identify a subset of genes whose misexpression is first observed in early generations, a step we refer to as priming; their misexpression then further progresses in late generations, as animals reach sterility. Analysis of misregulated genes shows that down-regulation of germline genes, expression of somatic transcriptional programs, and desilencing of the X-chromosome are concurrent events leading to loss of germline identity in both early and late generations. Upregulation of transcription factor LIN-15B, the C/EBP homologue CEBP-1, and TGF- pathway components strongly contribute to loss of fertility, and RNAi inactivation of cebp-1 and TGF-/Smad signalling delays the onset of sterility, showing they individually contribute to maintenance of germ cell identity. Our approach therefore identifies genes and pathways whose misexpression actively contributes to the loss of germ cell fate. More generally, our data shows how loss of a chromatin regulator in one generation leads to transcriptional changes that are amplified over subsequent generations, ultimately leading to loss of appropriate cell fate.
Transgenerational Transmission of Anxiety and Working Memory Deficit Following Paternal Aspartame Exposure
• Sara Jones, Florida State University
The use of low-calorie sweeteners, such as aspartame as weight loss tools is highly prevalent. Although aspartame is considered safe for human consumption, it can interfere with monoamine neurotransmitter synthesis, potentially contributing to behavioral changes. We found that exposure of adult C57BL/6 mice to aspartame in drinking water (0.015%; equivalent to 50%, of FDA approved maximum daily intake for humans) produced anxiety-like phenotype by the 8th week of exposure and working memory deficit by the 12th week (one-way ANOVA; p<0.05). We bred male mice from the 12-week aspartame exposure group with unexposed females to produce the F1 generation. Both male and female F1 offspring showed anxiety-like phenotype as well as working memory deficit (two-way ANOVA; p<0.05). Thus, aspartame exposure at levels as low as 50% of the FDA recommended intake may produce adverse behavioral phenotypes in the exposed individuals and their descendants. When we bred male mice from the 8-week aspartame exposure group, the F1 offspring did not show either behavioral phenotype (two-way ANOVA; p>0.05). Thus, although 8-weeks of aspartame exposure produced behavioral changes in the fathers, the transgenerational transmission occurred with 12- but not 8-weeks of paternal exposure. Thus, a temporal dissociation occurred between aspartame’s effects on somatic tissues (i.e. brain) and transgenerational transmission. Therefore, the effects on somatic tissues although detrimental, may not presage transgenerational transmission. The latter likely requires epigenetic modification of germ cells separate from the changes in somatic cells.