NEUROENDOCRINE GENE REGULATION BY HOMEODOMAIN PROTEINS

同源域蛋白对神经内分泌基因的调节

• 批准号: 2876779
• 负责人: Michael Selmanoff
• 金额: $ 0.03万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2876779
• 关键词: DNA footprinting; cell line; estrogen receptors; gel mobility shift assay; genetic mapping; genetic regulation; genetic regulatory element; genetic transcription; neuroendocrine system; oxytocin; protein isoforms; reporter genes; transcription factor; transfection; transfection /expression vector; vasopressins

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.

同源结构域超家族的转录因子起主要作用， 在神经系统发育、分化和调节中的作用 系统 POU III类蛋白的成员（Brn-1、-2和-4）是 在大脑发育过程中广泛表达，但仅限于特定的 神经元细胞群在成人下丘脑，特别是， 大细胞催产素（OT）和加压素（VP）表达神经元 视上核（SON）和室旁核（PVN）， 小细胞PVN Brn-2基因的切除导致了 发育中的大细胞神经元迁移并分化成 成熟神经元 因此，纯合Brn-2（-/-）敲除小鼠 缺乏OT和VP产生神经元以及小细胞PVN 神经元，并没有垂体后叶高兴。 杂合（+/-）Brn-2敲除小鼠具有成比例的减少， OT和VP基因转录本，表明Brn-2表达水平 在大细胞神经元直接控制这些转录 神经内分泌基因 主要工作假设 研究表明，这些最近克隆的同源结构域蛋白 调节神经内分泌中OT和VP基因的表达 下丘脑建议的研究重点是以下具体问题： 目的：确定转染的转录作用。 而 PI在其研究中使用了一些分子技术（即， 单细胞定量原位杂交组织化学 早期基因mRNA诱导、RNase保护试验和CNS 反义寡核苷酸微输注），他想成为 在分子生物学及其技术方面更加容易， 创造性地研究这些生殖器官-神经内分泌基因 基因表达水平的相互作用。 拟议休假工作 在彼得·伯巴赫博士的实验室里， 脑同源结构域蛋白（脑-1，-2 和-4）对催产素（OT）和加压素（VP）基因的影响， 鉴定赋予这些POU作用的顺式作用元件， 在肿瘤细胞系中使用异源表达的III类蛋白， 在器官型细胞培养物中同源表达。 这项工作将 涉及启动子-报告基因的设计、构建和应用 OT和VP基因的5 ′和3 ′侧翼区的构建体， 通过磷酸钙沉淀、基因枪和/或 重组腺病毒感染神经元（Neuro2A细胞）和非神经元（Non 神经元肿瘤细胞系（293个人胚肾细胞、猴胚肾细胞、人胚肾细胞） 肾CV-1细胞、BHK细胞和P19 EC细胞），报告基因测定 （荧光素酶和β-半乳糖苷酶），DNA酶I足迹分析和 利用COS细胞的电泳迁移率变动分析（EMSA）， 体外翻译 这一经验应使PI能够很好地 随后进行创新细胞和分子分析， 这些生殖激素对THE、GAD和GnRH基因的调控 神经内分泌下丘脑。 脑同源结构域蛋白 Brn-1、Brn-2和Brn-4对OT和VP基因的作用，并鉴定其顺式作用 赋予这些POU III类蛋白作用的元件， 肿瘤细胞系中异源表达和同源表达 在器官型细胞培养中。 这项研究将利用启动子- OT的5 ′-和3 ′-侧翼区的报告基因构建体 和VP基因，将这些构建体瞬时转染到神经元中， 和非神经元肿瘤细胞系以及器官型细胞培养物， DNA酶I足迹分析和电泳迁移率变动分析 （EMSA）利用COS细胞和体外翻译。 这项研究将 进一步了解这些POU II类的作用， 同源结构域蛋白在OT和VP转录调控中的作用 下丘脑神经内分泌的基因表达。