Exposure of neonatal rodents to high levels of exogenous estrogen leads to uterine abnormalities and neoplastic and other pathologies in adulthood. Similar effects occur in women exposed to the synthetic estrogen diethylstilbestrol (DES) in utero, so determining effects of early estrogen exposure has clinical significance. Estrogen effects are mediated primarily by estrogen receptor 1 (ESR1). Most ESR1 is nuclear, but 5-10% is located in cell membranes. Epigenetic changes induced by early estrogen exposure may be major factors in ensuing pathologies. Mechanisms by which early estrogen exposure produces epigenetic abnormalities are unclear, but may involve signaling pathways mediated through membrane ESR1 (mESR1). The overall objective of this proposal is to establish roles of mESR1 and nuclear ESR1 (nESR1) in uterine epigenetic effects of early estrogen treatment. To accomplish this, we will use two transgenic mice: nuclear-only estrogen receptor (NOER) mice lacking mESR1 and membrane-only estrogen receptor (MOER) mice lacking nESR1, along with wild-type (WT) and Esr1 knockout (Esr1KO) controls.
In Aim 1, WT, NOER, MOER and Esr1KO females will be injected with DES [1 mg/kg; postnatal days (PND) 1-5] or vehicle. Uteri of PND 5 pups will examined for histone methylation and acetylation marks critically involved in epigenetic gene regulation, as well as the kinase pathway and expression of the catalytic subunit of the methyltransferase complex responsible for one histone methylation mark. Other experiments in Aim 1 will determine if epigenetic effects of DES are entirely mediated through ESR1, or could involve other estrogen receptors such as ESR2 or G protein-coupled estrogen receptor (GPER). Epigenetic effects are reversible, so in Aim 2 we will use chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) to determine permanent changes in histone modifications of individual estrogen-responsive genes in adult uteri of mice treated on PND 1-5, as in Aim 1. We will examine key methylation and acetylation sites across regulatory and non-regulatory regions of three estrogen-responsive genes for which histone modifications following neonatal estrogen treatment have been reported, and also look for hyperresponsiveness of these genes to estrogen in the adult as a consequence of neonatal DES treatment. Our hypothesis is that epigenetic effects of early estrogen exposure on histone methylation and acetylation sites, and hyperresponsiveness of target genes in WT and NOER mice, will obligatorily require mESR1, and be absent in mice expressing nESR1 but lacking mESR1 (NOER). In addition, we postulate that mESR1 by itself will be insufficient to mediate estrogen-induced epigenetic effects and that these effects will also require nESR1. Results of these experiments will increase our understanding of the roles of mESR1, and how this receptor interacts with nESR1 to regulate epigenetic changes. These results will also increase understanding of the mechanistic basis by which early estrogen exposure alters histone methylation and acetylation in target genes, and the cascade of signaling events regulating histone methylation; these results will have clinical signficance.
Developmental exposure to estrogenic compounds causes short-term as well as long-term alterations in the cells of the female reproductive system that persist into adulthood, and are associated with increases in adult reproductive pathologies in women. Estrogens act through estrogen receptors (ERs) located either in the cell nucleus or in the cell membrane, and they can produce permanent changes called epigenetic modifications, which affect gene activity without changing the DNA sequence. The goal of this research is to clarify the roles of membrane and nuclear ER in the epigenetic effects of estrogens, and we anticipate that this work will identify obligatory roles for membrane estrogen receptors in this process and have human clinical significance.
| Rosenfeld, Cheryl S; Cooke, Paul S (2018) Endocrine disruption through membrane estrogen receptors and novel pathways leading to rapid toxicological and epigenetic effects. J Steroid Biochem Mol Biol : |
| Hess, Rex A; Cooke, Paul S (2018) Estrogen in the male: a historical perspective. Biol Reprod 99:27-44 |
