TRANSCRIPTIONAL REGULATION OF LH BETA GENE EXPRESSION

LH Beta 基因表达的转录调控

• 批准号: 6521235
• 负责人: LISA M HALVORSON
• 金额: $ 1.74万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2002-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6521235
• 关键词: biological signal transduction; cell line; cyclic AMP; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; luteinizing hormone; posttranslational modifications; protein kinase A; protein protein interaction; reporter genes; second messengers; tissue /cell culture; transcription factor; transfection

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are critical modulators of gamete maturation and gonadal steroidogenesis. Recent evidence has demonstrated that the transcription factors, steroidogenic factor-1 (SF-1) and early growth response gene 1 (Egr-1), regulate expression of the LHbeta-subunit gene. The long-term objective of our work is to understand fully the transcription factors and cis-elements which modulate LHbeta gene expression, information which will provide insight into both normal and abnormal reproductive function. The Specific Aims of this proposal are: 1) to characterize the role of the bicoid-related transcription factor, Ptx1, in mediating tissue-specific LHbeta gene expression, 2) to define the physiologic importance of the LHbeta-Ptx1 cis-element(s) in primary gonadotropes in culture and in vivo, and 3) to identify the molecular mechanisms which mediate cAMP/PKA-induced stimulation of LHbeta gene promoter activity. In Aim 1, based on a report by Drouin and co-workers, we hypothesize that Ptx1 acts alone and in synergy with SF-1 to increase LHbeta promoter activity, as will be tested by electrophoretic mobility shift assay, transient transfection of immortalized cell lines, and generation of Ptx1 "knockdown" cell lines. In Aim 2A, primary pituitary cell cultures will be infected using a highly efficient, recombinant adenovirus approach in order to define the 5'flanking region which confers gonadotrope-specific expression. The defined gonadotrope-specific region will then be used in reporter constructs as either the wild-type sequence or with a mutation in the Ptx1-site(s) identified in Aim 1. We predict that the presence of a mutation in the Ptx1 site(s) will markedly blunt reporter expression in both cultured primary pituitary cells (Aim 2B) and transgenic mouse lines (Aim 2C), verifying the physiologic importance of Ptx1 to LHbeta gene expression. In Aim 3, we propose to test the hypothesis that cAMP/PKA-induced increases in LHbeta promoter activity are due to: A) PKA-regulated changes in SF-1 and/or Egr-1 gene expression or post-translational modifications, and/or B) putative AP-2 cis-element(s) in the LHbeta gene promoter sequence. By further characterizing factors which mediate tissue-specific and hormonally-regulated LHbeta gene expression, these studies will contribute to our understanding of normal reproductive physiology, as well as the pathophysiology of disorders including infertility, polycystic ovarian syndrome, hypothalamic hypogonadism, and premature and delayed puberty.

垂体促性腺激素、黄体生成素 (LH) 和卵泡刺激素 (FSH) 是配子成熟和性腺类固醇生成的关键调节剂。 最近的证据表明，转录因子类固醇生成因子 1 (SF-1) 和早期生长反应基因 1 (Egr-1) 调节 LHbeta 亚基基因的表达。 我们工作的长期目标是充分了解调节 LHbeta 基因表达的转录因子和顺式元件，这些信息将提供对正常和异常生殖功能的深入了解。 该提案的具体目标是：1) 表征 bicoid 相关转录因子 Ptx1 在介导组织特异性 LHbeta 基因表达中的作用，2) 定义 LHbeta-Ptx1 顺式元件的生理重要性) 在培养物和体内的初级促性腺激素中，以及 3) 鉴定介导 cAMP/PKA 诱导的 LHbeta 基因启动子活性刺激的分子机制。 在目标 1 中，根据 Drouin 及其同事的报告，我们假设 Ptx1 单独作用并与 SF-1 协同作用以增加 LHbeta 启动子活性，这将通过电泳迁移率变化测定、永生化细胞系的瞬时转染进行测试，以及 Ptx1“敲低”细胞系的生成。 在目标 2A 中，将使用高效重组腺病毒方法感染原代垂体细胞培养物，以确定赋予促性腺激素特异性表达的 5' 侧翼区域。 然后，定义的促性腺激素特异性区域将作为野生型序列或在目标 1 中鉴定的 Ptx1 位点中带有突变的报告基因构建体中使用。我们预测 Ptx1 位点中存在突变（ s) 将显着减弱培养的原代垂体细胞 (Aim 2B) 和转基因小鼠系 (Aim 2C) 中报告基因的表达，验证了 Ptx1 对LHbeta 基因表达。 在目标 3 中，我们建议检验以下假设：cAMP/PKA 诱导的 LHbeta 启动子活性增加是由于：A) PKA 调节的 SF-1 和/或 Egr-1 基因表达或翻译后修饰的变化，以及/或B) LHbeta基因启动子序列中推定的AP-2顺式元件。 通过进一步表征介导组织特异性和激素调节的 LHbeta 基因表达的因素，这些研究将有助于我们了解正常生殖生理学以及包括不孕症、多囊卵巢综合征、下丘脑性腺功能减退症、早产和延迟性早产在内的疾病的病理生理学。青春期。