NEUROENDOCRINE GENE REGULATION BY HOMEODOMAIN PROTEINS

同源结构域超家族的转录因子起主要作用 在神经发育、分化和调节中的作用 系统。POU III类蛋白(BRN-1、-2和-4)的成员是 在大脑发育过程中广泛表达，但仅限于特定的 成人下丘脑的神经元细胞群，尤其是 大细胞催产素和加压素表达神经元 视上核(SON)和室旁核(PVN)以及 小细胞室旁核。BRN-2基因的去除导致不能 发育大细胞神经元以迁移和分化为 成熟的神经元。因此，纯合子BRN-2(-/-)基因敲除小鼠 缺少产生催产素和VP的神经元以及细小细胞室旁核 神经元，并且没有垂体节的后叶。 杂合子(+/-)BRN-2基因敲除小鼠成比例减少 OT和VP基因转录本的表达水平，表明BRN-2的表达水平 在大细胞中，神经元直接控制这些基因的转录 神经内分泌基因。下面的主要工作假设 研究表明，这些最近克隆的同源结构域蛋白 神经内分泌中OT和VP基因表达的调控 下丘脑。拟开展的研究主要集中在以下几个方面 目的：确定转染物的转录作用。而当 PI在他的研究中利用了一些分子技术(即， 单细胞定量原位杂交组织化学 早期基因mRNA诱导、核糖核酸酶保护试验与中枢神经系统 反义寡核苷酸显微输注)，他想成为 在分子生物学及其技术方面更加灵活，以便 创造性地追求这些生殖激素-神经内分泌基因 基因表达水平上的相互作用。拟议的休假工作 彼得·伯巴赫博士的实验室将专注于确定 脑内同源结构域蛋白(Brain-1，-2)的转录作用 和-4)催产素(OT)和加压素(VP)基因和 确定赋予这些POU行动的顺应性作用元素 利用异源表达的III类蛋白在肿瘤细胞系和 在器官典型细胞培养中的同源表达。这项工作将 涉及启动子-报告基因的设计、构建和使用 OT和VP基因5‘和3’侧翼区的构建， 钙-磷酸盐沉淀法、生物法和或 重组腺病毒在神经元(Neuro2A细胞)和非神经细胞中的感染 神经性肿瘤细胞系(293个人胚胎肾细胞、猴 肾CV-1细胞、BHK细胞和P19 EC细胞)，报告基因检测 (荧光素酶和β-半乳糖苷酶)、DNase I足迹分析和 应用COS细胞和In的凝胶迁移率改变分析(EMSA) 体外翻译。这一经历应该会很好地将PI定位为 随后进行创新的细胞和分子分析 生殖激素对GAD和GnRH基因的调控 在神经内分泌的下丘脑。脑内同源域蛋白 在OT和VP基因上的BRN-1、-2和-4，并鉴定顺式作用基因 这些POU III类蛋白的作用元件使用 肿瘤细胞系中的异源表达和同源表达 在器官典型细胞培养中。这项研究将利用启动子- OT5‘和3’侧翼区的报告基因构建 和VP基因，将这些构建体瞬时导入神经元 和非神经元肿瘤细胞系，并转化为器官型细胞培养， DNA酶I足迹分析和电泳迁移率改变分析 (EMSA)利用COS细胞和体外翻译。这项研究将 进一步加深我们对这些POU II类的作用的理解 同源结构域蛋白在OT和VP转录调控中的作用 神经内分泌下丘脑的基因表达。

Transcription factors of the homeodomain superfamily play a major role in the development, differentiation and regulation of the nervous system. Members of the POU class III proteins (Brn-1, -2 and -4) are widely expressed during brain development but are restricted to specific neuronal cell groups in the adult hypothalamus, in particular, the magnocellular oxytocin (OT) and vasopressin (VP) expressing neurons of the supraoptic (SON) and paraventricular (PVN) nuclei, and of the parvocellular PVN. Ablation of the Brn-2 gene causes an inability of developing magnocellular neurons to migrate and differentiate into mature neurons. Consequently, homozygous Brn-2 (-/-) knockout mice lack OT- and VP-producing neurons as well as parvocellular PVN neurons, and have no posterior lobe of the pituitary glad. Heterozygous (+/-) Brn-2 knockout mice have a proportional reduction of OT and VP gene transcripts, indicating that Brn-2 expression levels in magnocellular neurons directly control the transcription of these neuroendocrine genes. The principal working hypothesis under investigation is that these recently cloned homeodomain proteins regulate OT and VP gene expression in the neuroendocrine hypothalamus. Proposed research is focused on the following specific aim: to determine the transcriptional action of the transfection. While the PI has utilized some molecular techniques in his research (i.e., single cell quantitative in situ hybridization histochemistry, immediate early gene mRNA induction, RNase protection assay and CNS antisense oligonucleotide microinfusion), he would like to become much more facile in molecular biology and its techniques in order to creatively pursue these reproductive hormone-neuroendocrine gene interactions at the level of gene expression. Proposed sabbatical work in the laboratory of Dr. Peter Burbach will focus on determining the transcriptional action of the brain homeodomain proteins (Brain-1, -2 and -4) on the oxytocin (OT) and vasopressin (VP) genes and identifying the cis-acting elements conferring the actions of these POU class III proteins using heterologous expression in tumor cell lines and homologous expression in organotypical cell cultures. This work will involve the design, construction and use of promoter-reporter gene constructs for the 5'- and 3'-flanking regions of the OT and VP genes, transfection by calcium-phosphate precipitation, biolistics, and or recombinant adenoviral infection in neuronal (Neuro2A cells) and non- neuronal tumor cell lines (293 human embryonal kidney cells, monkey kidney CV-1 cells, BHK cells and P19 EC cells), reporter gene assays (luciferase and beta-galactosidase), DNase I footprint analysis and electrophoretic mobility shift assay (EMSA) utilizing COS cells and in vitro translation. This experience should well position the PI to subsequently pursue an innovative cellular and molecular analysis of THE, GAD and GnRH gene regulation by these reproductive hormones in the neuroendocrine hypothalamus. Brain homeodomain proteins Brn-1, -2 and -4 on the OT and VP genes, and to identify the cis-acting elements conferring the actions of these POU class III proteins using heterologous expression in tumor cell lines and homologous expression in organotypical cell cultures. This research will utilize promoter- reporter gene constructs for the 5'- and 3'-flanking regions of the OT and VP genes, transient transfection of these constructs into neuronal and non-neuronal tumor cell lines and into organotypic cell cultures, DNase I footprint analysis and electrophoretic mobility shift assay (EMSA) utilizing COS cells and in vitro translation. This research will further our understanding of the role of these POU class II homeodomain proteins in the transcriptional regulation of OT and VP gene expression in the neuroendocrine hypothalamus.