TRANSCRIPTIONAL REGULATION OF THE GFAP GENE

GFAP 基因的转录调控

• 批准号: 3415217
• 负责人: JACOB SARID
• 金额: $ 18.28万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415217
• 关键词: DNA binding protein; DNA footprinting; alkaline phosphatase; astrocytes; cell differentiation; developmental genetics; gene deletion mutation; gene expression; genetic enhancer element; genetic library; genetic manipulation; genetic regulatory element; genetic transcription; glial fibrillary acidic protein; in situ hybridization; laboratory rat; migration inhibition factor; molecular cloning; neoplastic transformation; newborn animals; nucleic acid sequence; reporter genes; transcription factor; transfection

This proposal describes experiments that will characterize elements which regulate the astrocyte-specific expression of the glial fibrillary acidic protein (GFAP) gene. This gene encodes the major component of astrocyte intermediate filaments and is spatially and temporally regulated in the developing central nervous system. Astrocytes constitute nearly 40% of the CNS cell population and are responsible for structural, nutritional, developmental and biochemical support of the neurons. They also play an important role in CNS response to disease and injury and give rise to a significant portion of CNS tumors in adults. Understanding the mechanisms which regulate astrocyte growth and differentiation is important for future studies of CNS development and glial cell transformation. Transcriptional regulation of the GFAP gene can serve as a system for the identification of molecular signals which modulate glial cell differentiation: the gene is expressed upon differentiation of immature glial cells to mature astrocyte suggesting that factors regulating its transcription play a major role in astroglial differentiation. A 5'-flanking region of the gene was cloned land is able to direct glial-specific reporter gene expression in transfected cells. Transfection of deletion constructs revealed that a cell-specific regulatory region resides upstream of the gene promoter. Primer extension analysis verified transcriptional regulation of constructs in transfected cells. An upstream cis-acting element has been identified. Gel shift assays have identified glial-specific trans-acting factor which bind to the cis-acting element. DNA sequence analysis, coupled with DNase assays, will , locate and identify more precisely the sequence motifs which bind to cell-specific trans-acting factors. cDNA libraries was prepared from mRNA of transformed glial cells, and a similar library will be made from primary astrocytes. Sequence motifs which bind to cell-specific nuclear factors will be used for screening the cDNA expression libraries and isolating cDNA clones which encode DNA-binding proteins. These clones will be used for studies of their role in the process of astrocyte differentiation and oncogenic transformation.

该建议描述了将表征元素的实验 调节神经胶质原纤维的星形胶质细胞特异性表达 酸性蛋白（GFAP）基因。 该基因编码 星形胶质细胞中间细丝，在空间和时间上是 在发展中心神经系统中受到监管。 星形胶质细胞 占CNS细胞群的近40％，并负责 结构，营养，发育和生化支持 神经元。 它们在CNS对疾病的反应中也起着重要作用 受伤并引起成人中枢神经系统肿瘤的很大一部分。 了解调节星形胶质细胞生长的机制和 差异化对于CNS发展的未来研究和 神经胶质细胞转化。 GFAP基因的转录调控 可以用作识别分子信号的系统 调节神经胶质细胞分化：该基因在 未成熟的神经胶质细胞与成熟星形胶质细胞的分化表明 调节其转录的因素在星形胶质细胞中起主要作用 分化。 基因的5'边缘区域是克隆的土地是 能够在转染中引导神经胶质特异性报告基因表达 细胞。 缺失构建体的转染表明细胞特异性 调节区域位于基因启动子的上游。 底漆 扩展分析验证了构建体的转录调控 转染的细胞。 已经确定了上游的顺式作用元素。 凝胶转移测定已确定神经胶质特异性的跨作用因子，该因子 与顺式作用元件结合。 DNA序列分析，与 DNase分析，将更精确地定位和确定序列 结合细胞特异性跨作用因子的基序。 cDNA库 是从转化的神经胶质细胞和类似文库的mRNA制备的 将由主要星形胶质细胞制成。 结合的序列基序 细胞特异性核因子将用于筛选cDNA 表达文库和隔离cDNA克隆，编码DNA结合 蛋白质。这些克隆将用于研究其在 星形胶质细胞分化过程 和致癌转化。