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.

垂体性促性腺激素，黄体生成激素（LH）和卵泡刺激激素（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-SUBUNIT基因的表达。 我们工作的长期目标是完全了解调节LHBETA基因表达的转录因子和顺式元素，这些信息将提供对正常生殖功能和异常生殖功能的见解。 该提案的具体目的是：1）表征与双子相关的转录因子PTX1在介导组织特异性LHBETA基因表达中的作用，2）定义LHBETA-PTX1 cis-cis-元素在培养和3型型型gonadotropes中的生理重要性，并定义了cor erigartial erestiation in Cruital和3）。 CAMP/PKA诱导的LHBETA基因启动子活性的刺激。 在AIM 1中，根据Drouin和同事的报告，我们假设PTX1单独起作用，并与SF-1协同作用，以增加LHBETA启动子活性，这将通过电泳移动性转移测定法测试，通过电泳迁移分析，永生细胞系的短暂转染和PTX1的生成PTX1“敲除” Cell line。 在AIM 2a中，将使用高效的重组腺病毒方法感染原发性垂体细胞培养物，以定义赋予促性腺纤维特异性表达的5'Franking区域。 然后，定义的促性腺特异性区域将在报道构建体中用作野生型序列，或在目标1中鉴定出的PTX1位点中的突变。我们预测，PTX1位点突变的存在在PTX1位点中存在突变（S）在两种培养的基本垂体细胞（AIM 2B）中（AIM 2B）（AIM 2B）（AIM 2B）（AIM 2B）中表达显着的（s） PTX1对LHBETA基因表达的重要性。 在AIM 3中，我们建议检验以下假设：cAMP/PKA诱导的LHBETA启动子活性的增加是：a）a）pKA调节的SF-1和/或EGR-1基因表达或翻译后修饰，以及/或B）在LHBETA基因促进剂中假定的AP-2 CIS-元素。 通过进一步表征介导组织特异性和荷尔蒙调节的LHBETA基因表达的因素，这些研究将有助于我们对正常生殖生理学的理解，以及疾病的病理生理学，包括不育，多囊性卵巢综合征，下丘脑下丘脑降低和延迟的次生和延迟的人。