Transcriptional Regulation of Keratocan and Mimecan

描述（由申请人提供）：基因的转录控制， 显示组织特异性表达被认为是重要的， 积极和消极调节因素的贡献是特别重要的 兴趣角蛋白聚糖和mimecan是合成的主要蛋白质， 蛋白聚糖在脊椎动物眼睛的角膜基质，但也不是 在其他组织中合成或部分糖基化合成 糖蛋白牛和人角膜蛋白聚糖基因的克隆及牛角膜蛋白聚糖的研究进展 mimecan基因，以及体外维持系统的可用性 和抑制这些基因的表达，提供了一个独特的模型， 研究它们的转录调控。本报告的总体目标 项目是分析顺式作用的转录调控元件， 角膜蛋白聚糖和mimecan基因，以鉴定与角膜蛋白聚糖相互作用的角膜蛋白。 这些元素，并测试这两个基因中的特定基因突变。的 以下问题将得到解决： mimecan和keratocan在眼组织与非眼组织中的受控性？哪些主题 角蛋白聚糖和mimecan转录调控区的表达解释了 血清和FGF-2对其表达的影响？什么转录因子 调节这些基因的组织特异性表达？有那些功能 在同一组织中合成的多种mimecan mRNA转录本是什么 多个顺式作用调节/定位元件内的顺式作用调节/定位元件的序列 mimecan mRNA转录物和什么蛋白质结合这些元素？实验 在体外进行的包括：DNA酶I超敏位点作图，荧光素酶 报告基因测定，单个元件的定点诱变/缺失 以确定其对功能的贡献，以及 新的转录因子的克隆和测序， 调控基序来表征它们在驱动转录中的作用。给定 硫酸角质素蛋白聚糖在提供角膜 透明度，这里提出的研究可能会提供新的信息， 角膜特异性转录调控，从而提出替代 角膜KSPGs治疗性上调的靶点 situations.此外，关于组织特异性转录的知识 成功地在体外产生组织， 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.