LAMININ GENE EXPRESSION IN GLOMERULAR CELLS

描述(改编自调查人员摘要)：这是一个修改后的 竞争续签申请，由经验丰富的 备受尊敬的调查员。LAMC1基因编码层粘连蛋白Gamma1 (前身为层粘连蛋白B2)，是异三聚体层粘连蛋白的一种成分， 存在于正常基底膜中并积聚的分子 在肾小球硬化方面。在过去的工作中，PI已经表明IL-1和TGF-1- B和PMA激活层粘连蛋白Gamma1基因在大鼠肾小球的表达 上皮细胞。他已经证明了核因子-kB在这一过程中的作用。 最近将这项工作扩展到一种新的转录 因素。比较人、大鼠和小鼠的5‘端基因组序列 他发现了一个新的、保守的序列，称为 BCN-1。他已经证明了这个序列结合了一个或多个核因子 系膜细胞经PMA或IL-1b处理后表达上调。这个 BCN-1序列存在于其他启动子(补体、胰岛素)和 BCN-1核因子也存在于猴肾COS-7细胞中 和人类Jurkat细胞。 他提出了三个目标：1.表征转录因子 结合BCN-1基序。使用酵母一号杂交系统，他已经 确定了几个因素(RTEF/EFT、TEF-3和两个部分小说 CDNA、K53和K46)。在酵母中，K53特别令人感兴趣，因为它 与完整但未突变的BCN-1序列结合(与RTEF和K46不同 这将两者捆绑在一起)。在系膜细胞中，TEF3和K46都激活了 带有完整的BCN-1位点但没有突变的BCN-1的LAMC1启动子 序列(未提供K53的完整cDNA)。自BCN-1活动以来 可以驻留在单个组件所缺少的复合体中 为了提高活性，酵母杂交系统可能会给出误导结果。他会的 因此，采取平行战略，将核抽提物分成 亲和DNA柱，并进行微测序。表征 BCN-1蛋白的研究将包括抗体产生、大小比较 重组产物与核提取蛋白之间凝胶位移的关系 分析，分析DNA结合的结构域，转录 激活和核定位信号。2.鉴定LAMC1内含子 对肾小球治疗有反应的因素和因素 IL-1b、转化生长因子-b和PMA阳性细胞。这些研究的基本原理是 来自PI实验室的数据表明，激动剂对mRNA的刺激 表达大于由于激动剂作用而表现出来的 发起人。虽然承认提高消息稳定性可以 考虑到这些观察结果，他建议更多的顺位行动 调控元件可能位于大鼠LAMC1的第一内含子 吉恩。因此，他将在老鼠体内寻找DNA酶敏感部位 吉恩。如果这些站点看起来与飞利浦已经发布的站点相似 和同事在小鼠内含子上，他将使用小鼠克隆提供 菲利普斯博士将基因转录提高了20倍，并在 小鼠肾小球细胞。然后，他将在数据库中搜索站点 并进行缺失分析，从已知的凝胶位移中寻找证据 转录因子结合到这些位点，并决定是否 增强子与启动子协同作用。3.定义转录 LAMC15‘-侧翼区中的沉默元件。在前期工作中， PI发现-233/-15区域有消音器。他会的 尝试进一步定位作用，进行体外DNA酶 足迹，并用转录中识别的序列进行搜索 因素数据库。如果消音器能缩小到10-20个基点， 则可以在-233/-15片段的上下文中进行突变以 取消消音器活动。他还将测试消声器的效果 异源启动子(SV40)。

DESCRIPTION (Adapted from Investigator's Abstract): This is an amended application for competitive renewal, submitted by an experienced and highly-respected investigator. The LAMC1 gene encodes laminin gamma1 (formerly laminin B2) that is a component of heterotrimeric laminin, a molecule that is present in normal basement membranes and accumulates in glomerulosclerosis. In past work, the PI has shown that IL-1 and TGF- b, and PMA activate laminin gamma1 gene expression in rat glomerular epithelial cells. He has shown that NF-kB contributes to this process and has recently extended this line of work to a novel transcriptional factor. In comparing the 5' genomic sequence of human, rat, and mouse laminin gamma1 he discovered a novel and conserved sequence, termed bcn-1. He has shown that this sequence binds a nuclear factor or factors which are upregulated by PMA or IL-1b treatment of mesangial cells. The bcn-1 sequence is present in other promoters (complement, insulin) and the BCN-1 nuclear factor is also present in monkey kidney COS-7 cells and human Jurkat cells. He proposes three aims: 1. To characterize transcription factors which bind to the bcn-1 motif. Using the yeast one hybrid system, he has identified several factors (RTEF/EFT, TEF-3, and two partial novel cDNAs, K53 and K46). In yeast, K53 is of particular interest because it binds to an intact but not mutated bcn-1 sequence (unlike RTEF and K46 which bind both). In mesangial cells, both TEF3 and K46 activate the LAMC1 promoter with an intact bcn-1 site but not with a mutated bcn-1 sequence (the full cDNA for K53 is not available). Since BCN-1 activity may reside within a complex of which the individual components lack activity, the yeast hybrid system may give misleading results. He will therefore pursue a parallel strategy to fractionate nuclear extracts on an affinity DNA column and carry out microsequencing. Characterization of BCN-1 protein will include antibody production, comparison of size between recombinant product and nuclear extract protein on gel shift analysis, analysis of domains for DNA binding, transcriptional activation, and nuclear localization signal. 2. To identify LAMC1 intron elements and factors that are responsive to treatment of glomerular cells with IL-1b, TGF-b, and PMA. The rationale for these studies is data from the PI's laboratory that agonist stimulation of mRNA expression is greater than what can be shown due to agonist effect on the promoter. While acknowledging that increased message stability could account for these observations, he proposes that additional cis acting regulatory elements may be located in the first intron of the rat LAMC1 gene. He will therefore search for DNase hypersensitive sites in the rat gene. If the sites appear similar to those already published by Philips and colleagues in the mouse intron, he will use mouse clones provided by Dr. Phillips that boost gene transcription 20 fold, and test them in mouse glomerular cells. He will then search for sites in the data base and carry out deletion analysis, seek evidence from gel shift that known transcription factors bind to these sites, and determine whether the enhancer synergizes with the promoter. 3. To define transcriptional silencing elements in the LAMC1 5'-flanking region. In preliminary work, the PI has found suggesting a silencer in region -233/-15. He will attempt further localize the effect, carry out in vitro DNase footprinting, and search with the identified sequence in transcription factor databases. If the silencer can been narrowed down to 10-20 bp, then mutations can be made in the context of the -233/-15 fragment to abolish silencer activity. He will also test the silencer effect on heterologous promoters (SV40).