TRANSCRIPTIONAL REGULATION OF COLLAGEN IV GENES

基底膜是由以下组成的细胞外膜结构 主要为IV型胶原、层粘连蛋白、尼德原和硫酸肝素蛋白多糖 成分，并由上皮(或内皮)在界面沉积 到空隙里。基底膜在体内起着至关重要的作用 正常胚胎发育、组织重塑(会愈合)和滤过 进程。它们还参与了疤痕形成等过程， 毛细血管和肾小球基底膜增厚和功能障碍 与年龄和糖尿病的情况，以及肝和角膜纤维化。阿尔 这些异常中的一种可以归因于 基底膜。 这项提议的长期目标是阐明 体内外基底膜的形成和维持， 特别关注组织特异性转录调控 IV型胶原基因。最近在这方面取得了进展 目的鉴定几种转录增强子 小鼠IV型胶原基因在COL4a1第一内含子和在 COL4a2第三内含子。第一内含子中的增强子之一(0.3L) COL4a1基因，已被鉴定并发现仅在 产生IV型胶原的细胞。它也被发现受到 F9细胞系统中的一种分化诱导方式，它模拟了 早期的老鼠发育。此外，0.3L的两个GAACAAT元素 增强子，似乎是新的顺式作用元件，是必不可少的 0.3L增强子的组织特异性活性。另一方面， COL4a2第三内含子增强子还没有被深入表征。 这项建议是专门为了获得对完成的支持 COL4a2第三内含子增强子的特征。冲向 这一目标建议的具体目标是：1)描述 转录增强的顺式作用元件 COL4a2第三内含子的活性，以及2)鉴定、纯化和 克隆与顺式作用元件结合的反式作用因子 小鼠COL4a2第三内含子增强子。这些目标将通过以下方式实现 缺失分析、突变等分子生物学技术 分析、转基因、分子克隆以及生化 蛋白质的提纯方法。

Basement membranes are extracellular membranes structures consisting of collagen IV, laminin, nidogen and heparan sulfate proteoglycan as major components and are deposited by epithelium (or endothelium) at interfaces to the interstitium. The basement membranes play essential roles in normal embryogenesis, tissue remodeling (would healing), and filtration process. They are also involved in such processes as scar formation, thickening and dysfunction of capillary and glomerular basement membrane with age and diabetic condition, and fibrosis in liver and cornea. Al of these abnormalities can be attributed to the malformation of the basement membrane. The long range goal of this proposal is to elucidate the mechanism for the formation and maintenance of basement membrane in vivo and in vitro, with a special focus on the tissue specific transcriptional regulation of collagen IV genes. Progress has been made recently toward this objective with the identification of several transcriptional enhancers of the mouse collagen IV genes in the COL4a1 first intron and in the COL4a2 third intron. One of the enhancers (0.3L), in the first intron of COL4a1 gene, has been characterized and found to function only in collagen IV producing cells. It has also been found to be regulated in a differentiation induced manner in the F9 cell system which models the early mouse development. Furthermore, two GAACAAT elements of the 0.3L enhancer, which appear to be new cis-acting elements, are essential for the tissue specific activity of the 0.3L enhancer. On the other hand the COL4a2 third intron enhancers have not been characterized in depth. This proposal is specially intended to obtain support for the completion of the characterization of the COL4a2 third intron enhancers. Toward this objective the proposed specific aims are: 1) To characterize the cis-acting elements responsible for the transcriptional enhancing activity of the COL4a2 third intron, and 2) To characterize, purify and clone the trans-acting factors that bind to the cis-acting elements of the mouse COL4a2 third intron enhancer. These aims will be pursued using molecular biological technique such as deletion analysis, mutational analysis, transfection, and molecular cloning as well as biochemical approach for protein purification.