Deciphering the molecular function of the estrogen receptor a in fat metabolism

繁殖是一个消耗大量能量的过程，对脂肪代谢有着深远的影响，而脂肪代谢是野生动物和家畜的主要能量来源。在哺乳动物中，卵巢性激素17b-雌二醇（E2）在控制生殖功能和调节脂质和葡萄糖稳态方面发挥着重要作用。例如，小鼠卵巢切除术导致脂质储存的显著重新分配和脂肪量的增加。相反，E2治疗可以恢复生殖缺陷小鼠的正常脂肪代谢。E2在脂肪组织中的这些作用是由雌激素受体a （ERa）介导的，雌激素受体a是一种在脂肪和生殖组织中均表达的配体依赖性转录因子。有趣的是，许多环境污染物（称为内分泌干扰化学物质（EDCs））有可能作为ERa激动剂或拮抗剂，潜在地影响脂肪代谢。另一方面，脂肪组织也通过分泌脂肪因子等生物活性肽，在生殖轴的调控中发挥重要作用。因此，E2信号通路在生殖功能和脂肪代谢之间的冗余性对于理解能量调节和生殖适应性之间的相互作用具有重要意义。本研究计划的主要目的是建立ERa在脂肪代谢中的作用，并探讨E2和雌激素和抗雌激素EDCs调节脂肪组织基因表达的机制。我们将验证ERa是指导脂肪细胞类型特异性基因表达程序和生殖周期中脂肪细胞功能调节剂的重要调节因子的假设。两个具体目标是：(1)探索雌激素在发情周期脂肪组织中特异性转录程序的建立中的作用；(2)研究具有雌激素和抗雌激素活性的EDCs对脂肪组织中雌激素信号传导的影响。我们将首先探索ERa耗尽雌性小鼠与野生型（WT）小鼠相比脂肪组织的形态学和关键生理特征。我们将使用RNA-seq技术对小鼠发情周期中脂肪组织中ERa缺失的影响进行全基因组转录组学分析。随后将进行ChIP-seq分析，以确定WT脂肪组织中的ERa结合位点。对于第二个目标，我们将确定特定的EDCs是否干扰脂肪组织中的ERa信号，以及这如何影响脂肪组织的表型。我们将首先利用小鼠模型评估不同的EDCs对脂肪组织中ERa活性的影响，该模型在含有雌激素反应元件（ERE）的启动子控制下内源性表达荧光素酶报告基因。通过对脂肪组织进行更深入的生理和分子分析，将进一步研究发现的调节ERa功能的EDCs。这些实验将包括脂肪细胞的体视学检查和内分泌参数的具体测量。此外，我们还将利用RT-qPCR和ChIP技术在脂肪细胞中研究EDCs对目的1中鉴定的ERa靶基因调控的分子作用。总之，我们提出了一项新颖而全面的研究，使用多种模型和技术来确定E2等生殖激素调节动物脂肪代谢的分子机制，以及EDCs如何干扰这一生理途径以影响生殖健康。

Reproduction is an energetically costly process with profound effects on fat metabolism, the major form of energy source in wild and domestic animals. In mammals, the ovarian sex hormone 17b-estradiol (E2) has major roles in both the control of reproductive functions and in the regulation of lipid and glucose homeostasis. For example, ovariectomy in mice causes a significant redistribution of lipid storage and an increase in fat mass. Conversely, E2 treatment restores normal fat metabolism in mice with reproductive deficiency. These effects of E2 in fat tissues are mediated by the oestrogen receptor a (ERa), a ligand-dependent transcription factor expressed in both adipose and reproductive tissues. Interestingly, many environnemental contaminants (called endocrine disrupting chemicals (EDCs)) have the potential to act as ERa agonists or antagonists, potentially affecting fat metabolism. On the other hand, adipose tissues also play an important role in the regulation of the reproductive axis by secreting bioactive peptides such as adipokines. Thus, the redundancy in E2 signalling pathways between reproductive functions and fat metabolism are of major interest for the understanding of the interplay between energy regulation and reproductive fitness. The main objective of this research program is to establish the role of ERa in fat metabolism and explore the mechanisms by which E2 and estrogenic and antiestrogenic EDCs regulates gene expression in adipose tissues. We will test the hypothesis that ERa is an essential regulator in directing adipose cell-type-specific gene expression program and a modulator of fat cell function during the reproductive cycle. The two specific objectives are: (1) to explore the role of ERa in the establishment of specific transcriptional programs in adipose tissue during estrous cycle, and (2) to examine the impact of EDCs with estrogenic and anti-estrogenic activity on ERa signalling in fat tissues. We will first explore the morphology and determine the key physiological features of the adipose tissue in ERa depleted female mice as compared to their wild-type (WT) counterpart. We will undertake genome-wide transcriptomic analysis of the effects of ERa deletion in the adipose tissue during the estrous cycle in mice using RNA-seq technology. This will be followed by ChIP-seq analysis to identify the ERa binding sites in WT adipose tissue. For the second objective, we will determine whether specifics EDCs interfere with ERa signalling in fat tissues and how this affect adipose tissues phenotype. We will first evaluate the impact of different EDCs on ERa activity in adipose tissue using a mouse model that endogenously express the luciferase reporter gene under the control of a promoter containing an estrogen responsive element (ERE). EDCs found to modulate ERa function will be further investigated with a more in-depth physiological and molecular analysis of fat tissue. These experiments will include stereological examination of the adipocytes and specific measurements of endocrine parameters. Moreover, we will also investigate the molecular effects of EDCs on the regulation of ERa target genes identified in the objective 1, using RT-qPCR and ChIP assays in adipocytes. Overall, we propose a novel and comprehensive investigation, using multiple models and techniques to determine the molecular mechanisms by which a reproductive hormone such as E2 regulates fat metabolism in animals and how EDCs may interfere in this physiological pathway to affect reproductive fitness.