Transcriptional Regulation of Keratocan and Mimecan

描述(申请人提供)：对符合以下条件的基因进行转录控制 展示组织特异性表达被认为是重要的，并且 积极的和消极的监管因素的贡献尤其重要 利息。角化蛋白和粘蛋白是主要合成的蛋白质。 脊椎动物眼睛角膜基质中的蛋白多糖，但要么不是 在其他组织中合成或部分糖基化合成 糖蛋白。牛和人角化蛋白聚糖基因及牛的最新克隆 Mimecan基因，以及可用于维护的体外系统 并抑制这些基因的表达，为 这样做的总体目标是 项目是分析顺式作用的转录调控元件 角质形成蛋白和粘附素基因，以识别与之相互作用的角膜蛋白 这些元素，并测试这两个基因的特定基因突变。这个 以下问题将被解决：转录速度如何 在眼组织和非眼组织中控制的MIMECAN和KERATON？哪些主题 角质形成蛋白和粘附素转录调控区域的 血清和成纤维细胞生长因子-2对其表达的影响？哪些转录因子 调控这些基因的组织特异性表达？有哪些功能？ 在同一组织中合成的多个Mimecan mRNA转录本？是什么 顺式作用调控/定位元件序列在多个 Mimecan mRNA转录本和哪些蛋白质与这些元件结合？实验 在体外进行的操作包括：DNase I超敏部位定位，荧光素酶 报告基因分析、单个元件的定点突变/缺失 来自监管区域以确定其对职能的贡献，以及 结合蛋白的新转录因子的克隆和测序 调控基序，以表征它们在驱动转录中的作用。vt.给出 硫酸角蛋白多糖在角膜供应中的重要性 透明度，这里提出的研究可能会提供关于 角膜特异性转录调控，从而提出了替代方案 治疗性上调角膜KSPGs的病理靶点 情况。此外，关于组织特异性转录的知识 为了成功地体外产生组织，需要进行调节 移植。

DESCRIPTION (provided by applicant): Transcriptional control for genes which exhibit tissue-specific expression is recognized to be important, and the contribution by positive and negative regulatory elements is of particular interest. Keratocan and mimecan are proteins that are synthesized as major proteoglycans in the corneal stroma of the vertebrate eye, but are either not synthesized in other tissues or are synthesized as partially glycosylated glycoproteins. Recent cloning of bovine and human keratocan genes and bovine mimecan gene, as well as the availability of an in vitro system for maintenance and suppression of expression of these genes, provide a unique model for studying their transcriptional regulation. The overall objective of this project is to analyze the cis-acting transcriptional regulatory elements of keratocan and mimecan genes, to identify corneal proteins that interact with these elements, and to test for specific genetic mutations in both genes. The following questions will be addressed: How are the transcription rates of mimecan and keratocan controlled in ocular vs. non-ocular tissues? Which motifs of the keratocan and mimecan transcriptional regulatory regions account for the serum and FGF-2 effects on their expression? What transcription factors regulate the tissue-specific expression of these genes? What are the functions of multiple mimecan mRNA transcripts synthesized in the same tissue? What is the sequence of cis-acting regulatory/localization elements within the multiple mimecan mRNA transcripts and what proteins bind to these elements? Experiments performed in vitro include: DNase I hypersensitive site mapping, luciferase reporter gene assays, site-directed mutagenesis/deletion of individual elements from the regulatory region to determine their contributions to function, and the cloning and sequencing of new transcription factors that bind to the regulatory motif to characterize their roles in driving transcription. Given the importance of keratan sulfate proteoglycans in providing corneal transparency, the research proposed here may provide novel information about cornea-specific transcriptional regulation and thereby suggest alternative targets for therapeutic up-regulation of corneal KSPGs in pathological situations. In addition, knowledge about tissue-specific transcriptional regulation will be required for successful in vitro generation of tissues for transplantation.