Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
Department of Biochemistry, School of Medicine, Fujita Health University, Toyoake, Japan.
Front Endocrinol (Lausanne). 2020 Jun 3;11:306. doi: 10.3389/fendo.2020.00306. eCollection 2020.
An important function of aromatase in the brain is conversion of testosterone secreted from the testis into estradiol. Estradiol produced in the brain is thought to be deeply involved in the formation of sexually dimorphic nuclei and sexual behavior as a neurosteroid. We analyzed the brain-specific promoter to elucidate the control mechanisms of brain aromatase expression that may be highly involved in sexual differentiation of the brain. The 202-bp upstream region of the brain-specific exon 1 has three types of -acting elements, aro-AI, AII, and B. We isolated ARP-1 as an aro-AII-binding protein by yeast one-hybrid screening from a cDNA library of mouse fetal brains. ARP-1 is a member of the nuclear receptor superfamily and functions as an orphan-type transcription factor. ARP-1 protein synthesized showed the same binding property to the aro-AII site as nuclear extract from fetal brains. To determine how the promoter is involved in brain-specific transcription of the aromatase gene, we first detected the occupancy of the aro-AII site by ARP-1 using chromatin immunoprecipitation assays. Diencephalic regions of fetal brains at embryonic day 16 were analyzed, which revealed ARP-1 recruitment to the aro-AII site. To analyze the effects of ARP-1 on transcriptional regulation of the brain-specific aromatase promoter, a luciferase reporter plasmid driven by the brain-specific promoter was transfected into CV-1 cells together with a plasmid expressing ARP-1 protein. These analyses revealed that ARP-1 induced promoter activity in a dose-dependent manner. Furthermore, to determine whether ARP-1 is required for aromatase expression in neurons, ARP-1 knockdown was conducted in neuronal cell primary culture. Knockdown of ARP-1 significantly suppressed the increase in aromatase mRNA observed in cultured neurons. These results indicate that ARP-1 is involved in the transcriptional regulation of the brain-specific promoter of the aromatase gene.
芳香化酶在大脑中的一个重要功能是将睾丸分泌的睾酮转化为雌二醇。大脑中产生的雌二醇被认为作为神经甾体，深度参与性二态性核和性行为的形成。我们分析了脑特异性启动子，以阐明可能高度参与大脑性分化的脑芳香化酶表达的控制机制。脑特异性外显子 1 的 202bp 上游区域具有三种类型的 -作用元件，即 aro-AI、AII 和 B。我们通过酵母单杂交筛选从小鼠胎脑 cDNA 文库中分离出 ARP-1 作为 aro-AII 结合蛋白。ARP-1 是核受体超家族的成员，作为孤儿型转录因子发挥作用。合成的 ARP-1 蛋白显示出与胎脑核提取物相同的 aro-AII 位点结合特性。为了确定启动子如何参与芳香化酶基因的脑特异性转录，我们首先使用染色质免疫沉淀测定法检测 ARP-1 对 aro-AII 位点的 占有率。分析了胚胎期 16 天的胎脑间脑区域，结果显示 ARP-1 募集到 aro-AII 位点。为了分析 ARP-1 对脑特异性芳香化酶启动子转录调控的影响，将由脑特异性启动子驱动的荧光素酶报告质粒与表达 ARP-1 蛋白的质粒共转染 CV-1 细胞。这些分析表明，ARP-1 以剂量依赖的方式诱导启动子活性。此外，为了确定 ARP-1 是否是神经元中芳香化酶表达所必需的，在神经元原代培养物中进行了 ARP-1 敲低。ARP-1 敲低显著抑制了培养神经元中观察到的芳香化酶 mRNA 的增加。这些结果表明 ARP-1 参与了脑特异性芳香化酶基因启动子的转录调控。
