Recent studies suggest that in utero exposure to environmental toxins can produce phenotypic changes over several successive generations. Although initial work suggests that epimutations propagate these transgenerational phenotypes, the cells that must carry this information to the next generation have not been examined. Primordial germ cells (PGCs), the embryonic founders of the egg or sperm, undergo rapid epigenetic reprogramming during mid-gestation in a timeframe that overlaps with the observed sensitivity to transgenerational effects following exposure to certain toxins. The long-term goal of our research is to elucidate genetic and epigenetic mechanisms underlying PGC development. The objective of this application is to assess the impact of one class of environmental chemical, phthalates, on mouse PGC development and epigenetic reprogramming in the exposed individual (F1) and subsequent generations (F3). Our central hypothesis is that biologically relevant doses of phthalates disrupt DNA methylation changes involved in reprogramming of PGCs during development, leading to gene expression changes; in order to be heritable through generations, it follows that mismethylation of regions associated with the regulation of reprogramming in PGCs disrupts expression of critical reprogramming machinery in the germline. This hypothesis is based upon prior reports of transgenerational phenotypes produced by a mixture of plasticizers BPA and phthalates as well as the demonstration that phthalates alter DNA methylation at the imprinted loci in humans and mice. Our hypothesis will be tested with the following two specific aims: (1) to establish phenotypic consequences of phthalate exposure on F1 and F3 generation mouse PGCs and (2) to determine direct and transgenerational effects of phthalates on the mouse PGC epigenome and transcriptome. In the first aim, we will interrogate PGC phenotypes in the F1 and F3 generation following in utero exposure to a range of environmentally relevant doses of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). Doses producing phenotypes may be informative for the second aim, in which we will survey epigenetic reprogramming in F1 and F3 PGCs by examining histone modifications and global methylation semi-quantitatively and by genome-wide methylation analysis. Resulting changes in the F1 and F3 PGC transcriptome, together with the epigenetic data, will reveal the consequences of observed dysregulation at the level of DNA methylation, enable prediction of phenotypes in other tissues, and could identify candidate epigenetic regulators responsible for propagating observed epimutations. The proposed work is innovative as it uses advanced, genome-wide approaches and, apart from previous work, will directly examine epigenetic changes and potential consequences in the very cell responsible for trangenerational transmission. The proposed work is significant as it will determine the extent to which phthalate exposure alters reprogramming in PGCs and potentially identify candidate mechanisms of transgenerational epigenetic inheritance.
Since phthalates are detectable in 100% of pregnant women in a recent U.S. cohort, the proposed studies are relevant to public health because the extent to which this ubiquitous class of plasticizing chemical affects epigenetic reprogramming in the germ cells-whether heritably or not- will provide important information on risk to future generations, in the worst scenario, or in the best-case help define risks to fertility of in utero exposed individuals. Defining the epigenetic changes induced by phthalate exposure to the cells that transmit information between generations will ultimately inform decisions by consumers, manufacturers and public health agencies.
