TRANSCRIPTIONAL REGULATION OF COLLAGEN IV GENES

基底膜是细胞外膜结构， IV型胶原、层粘连蛋白、巢蛋白和硫酸乙酰肝素蛋白聚糖是主要的 成分，并由上皮（或内皮）沉积在界面处 去找氚 基底膜起着至关重要的作用， 正常胚胎发生、组织重塑（伤口愈合）和滤过 过程 它们也参与疤痕形成， 毛细血管和肾小球基底膜增厚和功能障碍 以及肝和角膜纤维化。 Al 这些异常可以归因于畸形的 基底膜 本提案的长期目标是阐明 在体内和体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 小鼠胶原IV基因在COL4a1第一内含子和在 COL4a2第三内含子。 其中一个增强子（0.3L），位于第一个内含子中 COL4a1基因，已被表征并发现仅在 产生IV型胶原的细胞。 它也被发现是受管制的， F9细胞系统中的分化诱导方式，其模拟了 小鼠早期发育 此外，0.3L的两个GAACAAT元件 增强子，这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第三内含子增强子尚未深入表征。 这一建议特别是为了获得支持， COL4a2第三内含子增强子的表征。 朝向 为了实现这一目标，建议的具体目标是：1）描述 负责转录增强的顺式作用元件 C0L4a2第三内含子的活性，和2）为了表征、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： 分子生物学技术，如缺失分析、突变分析 分析、转染、分子克隆以及生化 蛋白质纯化的方法。

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.