Membrane estrogen receptor 1 mediation of epigenetic effects of estrogen

项目摘要 新生啮齿动物暴露于高水平的外源性雌激素导致子宫异常和肿瘤 和其他的疾病类似的影响也发生在接触合成雌激素的女性身上 己烯雌酚（DES）在子宫内，因此确定早期雌激素暴露的影响具有临床意义。 雌激素的作用主要由雌激素受体1（ESR 1）介导。大多数ESR 1是核的，但5-10%是核的。 位于细胞膜中。早期雌激素暴露引起的表观遗传学变化可能是导致 随之而来的病理。早期雌激素暴露产生表观遗传异常的机制是 目前尚不清楚，但可能涉及通过膜ESR 1（mESR 1）介导的信号通路。整体 本提案的目的是确定mESR 1和核ESR 1（nESR 1）在子宫表观遗传中的作用， 早期雌激素治疗的效果。为了实现这一目标，我们将使用两只转基因小鼠：仅核雌激素 缺乏mESR 1的NOER受体（NOER）小鼠和缺乏nESR 1的仅膜雌激素受体（MOER）小鼠， 沿着野生型（WT）和Esr 1敲除（Esr 1 KO）对照。在目标1中，WT、NOER、MOER和Esr 1 KO 雌性动物将注射DES [1 mg/kg;出生后1-5天（PND）]或载体。PND 5幼仔的子宫将 检查组蛋白甲基化和乙酰化标记在表观遗传基因调控中的重要作用，以及 作为激酶途径和甲基转移酶复合物的催化亚基的表达， 一个组蛋白甲基化标记。目标1中的其他实验将确定DES的表观遗传效应是否完全 通过ESR 1介导，或可能涉及其他雌激素受体，如ESR 2或G蛋白偶联雌激素 GPER受体。表观遗传效应是可逆的，因此在目标2中，我们将使用染色质免疫沉淀 通过ChIP（ChIP）和定量PCR（qPCR）来确定个体细胞中组蛋白修饰的永久性变化。 在PND 1-5处理的小鼠的成年子宫中的雌激素应答基因，如在Aim 1中。我们将研究关键 甲基化和乙酰化位点的调控和非调控区的三个雌激素反应， 已经报道了新生儿雌激素治疗后组蛋白修饰的基因，并且还观察了 作为新生儿DES治疗的结果，这些基因对成人雌激素的高反应性。 我们的假设是，早期雌激素暴露对组蛋白甲基化和乙酰化的表观遗传学影响 WT和NOER小鼠中靶基因的位点和高反应性，将强制性地需要mESR 1， 在表达nESR 1但缺乏mESR 1的小鼠中不存在（NOER）。此外，我们假设mESR 1本身 将不足以介导雌激素诱导的表观遗传效应，这些效应也需要nESR 1。 这些实验的结果将增加我们对mESR 1的作用的理解，以及这种受体是如何在细胞中发挥作用的。 与nESR 1相互作用以调节表观遗传变化。这些结果也将增加对 早期雌激素暴露改变靶基因中组蛋白甲基化和乙酰化的机制基础， 以及调控组蛋白甲基化的级联信号事件，这些结果将具有临床意义。

Project Summary 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.