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会导致子宫异常和肿瘤 以及成年后的其他病症。在接触合成雌激素的女性身上也会出现类似的效果 子宫内注射己烯雌酚(DES)，因此测定早期雌激素暴露的效果具有临床意义。 雌激素效应主要由雌激素受体1(ESR1)介导。大多数ESR1是核能，但5%-10%是 位于细胞膜中。早期雌激素暴露引起的表观遗传学改变可能是高血压的主要因素 随之而来的病态反应。早期雌激素暴露导致表观遗传异常的机制是 不清楚，但可能涉及通过膜ESR1(MESR1)介导的信号通路。整体而言 本研究的目的是确定mESR1和核ESR1(NESR1)在子宫表观遗传学中的作用 早期雌激素治疗的效果。为了做到这一点，我们将使用两只转基因小鼠：仅含核的雌激素 缺乏mESR1的受体(NOER)小鼠和缺乏nESR1的膜型雌激素受体(MOER)小鼠， 以及野生型(WT)和ESR1基因敲除(Esr1KO)对照。在目标1中，WT、NOER、MOER和Esr1KO 雌性将注射DES[1毫克/公斤；出生后1-5天]或赋形剂。PND 5幼崽的子宫将 还检查了组蛋白甲基化和乙酰化标记，这些标记与表观遗传基因调控密切相关 作为激酶途径和甲基转移酶复合体催化亚单位的表达，负责 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ESR1介导，或可能涉及其他雌激素受体，如ESR2或G蛋白偶联雌激素 受体(GPER)。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 (芯片)和定量聚合酶链式反应(QPCR)以确定个体组蛋白修饰的永久性变化 PND 1-5处理的成年小鼠子宫中的雌激素反应基因，如目标1所示。 三个雌激素反应基因调控和非调控区域的甲基化和乙酰化位点 已经报道了新生儿雌激素治疗后组蛋白修饰的基因，也正在寻找 作为新生儿DES治疗的结果，这些基因在成人中对雌激素的高反应性。 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效应 在WT和NOER小鼠中，靶基因的位置和高反应性将强制性地需要mESR1，并被 在表达nESR1但缺乏mESR1(NOER)的小鼠中不存在。此外，我们假设mESR1本身 不足以调节雌激素诱导的表观遗传效应，这些效应也需要nESR1。 这些实验的结果将增加我们对mESR1的作用以及该受体如何 与nESR1相互作用，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 以及调节组蛋白甲基化的一系列信号事件；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.