Session 1 Abstracts

OPENING KEYNOTE                                               

Germ Cell Exposures and Heritable Effects: Is Our Regulatory Paradigm Failing to Protect Future Generations?

David M. DeMarini, Retired, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA

The biology of germ cells is distinctly different from that of somatic cells.  However, regulatory agencies worldwide evaluate agents almost only in somatic cell mutagenicity assays and assume that positive results identify potential germ cell mutagens. Eighty-five agents are proven germ cell mutagens in rodents, but no agents have been declared germ cell mutagens in humans. Nonetheless, growing evidence indicates that tobacco smoking, air pollution, and ionizing radiation are likely human germ cell mutagens. The safety of future generations would be improved by requiring regulatory testing for germ cell effects and by not replacing all animal testing with high-throughput testing.   

MECHANISMS OF INTERGENERATIONAL INHERITANCE, PART 1 

Integration of Epigenetic Mechanisms in the Germline and Embryo in Epigenetic Transgenerational Inheritance 

Michael K. Skinner

Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, Washington, USA

Epigenetic Transgenerational Inheritance effects of environmental toxicants, nutrition, or stress significantly amplify the biological impacts and health hazards of these exposures. One of the most sensitive periods to exposure is during fetal gonadal sex determination when the germ line is undergoing epigenetic reprogramming and DNA re-methylation occurs. Previous studies have shown that toxicants can cause an increase in adult onset disease such as infertility, prostate, ovary and kidney disease, cancers and obesity. Interestingly, this effect is transgenerational (F1, F2, F3 and F4 generations) and is due to permanently (imprinted-like) altered epimutations in the germline. A parent-of-origin allelic epigenetic contribution has also been observed in transgenerational inheritance. The transgenerational epigenetic mechanisms appear to involve the actions of an environmental exposure to permanently alter the epigenetic (e.g. DNA methylation) programming of the germline that then alters the epigenetics and transcriptomes of the developing embryo to impact all subsequent somatic cell types and developing organs to induce disease susceptibility and development transgenerationally. In addition to DNA methylation, alterations in sperm ncRNAs and histone retention and modifications have also been observed. Recently, we have found the integration of concurrent differential DNA methylation regions (DMRs), ncRNAs, and differential histone retention regions (DHRs) in the germline are coordinately regulated and often co-localized in the same chromosomal regions. Observations indicate that environmental exposures can reprogram and integrate the various epimutations in the germline to impact the early embryo epigenetics and transcriptomes to induce epigenetic transgenerational inheritance of disease and phenotypic variation. This provides a basic molecular mechanism for this non-genetic form of inheritance.

Mechanisms of Transgenerational Inheritance of Obesity Epiphenotypes

Victor Corces

Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA

Mechanisms by which epiphenotypes are transmitted between generations through the germline remain poorly understood. Most promoters in mouse sperm and oocytes contain RNAPII and are flanked by positioned nucleosomes marked by a variety of active histone modifications. The sperm genome is bound by transcription factors, including Mediator, FoxA1, ER1 and AR. These proteins are found at promoters, enhancers, and super-enhancers. CTCF and cohesin are also present in sperm DNA, where they mediate interactions that organize the sperm genome into domains overlapping extensively with those found in mESCs. These results suggest that epigenetic information present in mammalian germ cells could be altered by environmental factors to cause novel phenotypic outcomes in the next generation. When pregnant females are exposed to endocrine disruptor chemicals, their progeny show a variety of phenotypes, including obesity. This phenotype is transmitted from the F1 to F6 generations through both the male and female germlines in the absence of further exposure, but it disappears in the F7 generation. Approximately 12 new protein binding sites are present in the sperm of obese mice from the F1 through the F6 generations. These new binding sites correspond to CTCF, RA, and ER1, suggesting that effects of these proteins on 3D chromatin organization and transcription of specific genes are responsible for the establishment and transmission of epiphenotypes. Changes in CTCF/cohesin are accompanied by alterations in 3D organization that affect enhancer-promoter interactions. Comparison of the transmission of obesity with alterations in the binding of these transcription factors in both sperm and oocytes points to the activation of an enhancer in an intron of the Fto gene as the cause of transgenerational transmission of obesity. The results suggest that both genetic and epigenetic alterations of the same gene can lead to adverse effects on human health.

Blood Factors Are Vectors of Communication Between Exposure and the Germline

Isabelle M. Mansuy

Laboratory of Neuroepigenetics, University and ETH Zürich, Brain Research Institute,
Winterthurerstrasse 190, Zürich, Switzerland. Email: mansuy@hifo.uzh.ch

Environmental factors can change phenotypes in exposed individuals and their offspring across generations. When exposure is chronic and systemic, particularly in early life, all cells in the body including germ cells can be affected. The signals involved in the embedding of exposure into germ cells are not well understood. We used a mouse model of postnatal exposure to traumatic stress to study these signals. In humans, childhood trauma affects up to 25% of children worldwide and is known to cause mental disorders and chronic comorbidities across families. In the mouse model, we observed that postnatal trauma induces risk-taking behaviors, depressive-like symptoms and cognitive and social deficits in exposed males and their offspring, in some cases up to the 4th generation1–3. Metabolic functions and pathways, particularly lipid-derived metabolites, were also severely dysregulated. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed mouse males into controls recapitulated the metabolic phenotype in the offspring. We identified circulating factors involving peroxisome proliferator-activated receptor (PPAR) pathways in these effects and found that pharmacological PPAR activation in vivo reproduces the metabolic dysfunctions in the offspring and grand-offspring of injected males, and affects the sperm transcriptome in fathers and sons4. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.

Sperm-borne tsRNAs Are Required for Maternal Transmission of Epimutations

Wei Yan

Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA 90502, USA 

The molecular mechanism underlying epigenetic inheritance remains largely unknown. Recent studies have suggested that sperm-borne tRNA-derived small RNAs (tsRNAs) act as an epigenetic carrier essential for paternal transmission of acquired traits or disease phenotypes induced by unhealthy diet or environmental factors. DNMT2, as a methyltransferase responsible for the 5mC modification on C38 of three tRNAs, has been implicated in the transmission of paternal epigenetic phenotypes, e.g., a Kit paramutation-induced white tail tip phenotype and metabolic disorders induced by a high fat diet, through regulating C38 5mC contents in tRNAs and consequently tsRNA biogenesis in sperm. However, it remains unknown whether the sperm-borne, DNMT2-dependent tsRNAs are also required for maternal transmission (via oocytes) of non-genetic phenotypes and how these DNMT2-dependent tsRNAs function to influence the transmission of non-genetic phenotypes. Here, we show that DNMT2 is required for normal expression profiles of tsRNAs and other sncRNAs in sperm, and disrupted DNMT2-dependent tsRNA and sncRNA profiles due to Dnmt2 inactivation block maternal transmission of a Kit paramutation-induced white tail tip phenotype in mice. The sperm-borne, DNMT2-dependent tsRNAs and miRNAs tend to target genes involved in histone modifications and chromatin organization in the pronuclear stages of embryonic development. Our data reveal an essential role of sperm-borne DNMT2-dependent tsRNAs and other sncRNAs in efficient transmission of epigenetic phenotypes through the maternal germline.