Transcriptional Regulation of Keratocan and Mimecan

Keratocan 和 Mimecan 的转录调控

基本信息

批准号：
6759979
负责人：
GARY W CONRAD
金额：
$ 25.37万
依托单位：
KANSAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-01 至 2006-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：基因的转录控制表现出组织特异性表达很重要，并且正面和负调节元素的贡献特别是兴趣。角质和mimecan是合成为主要的蛋白质脊椎动物眼的角膜基质中的蛋白聚糖，但不是在其他组织中合成或合成为部分糖基化糖蛋白。牛和人类角质基因和牛的最新克隆 Mimecan基因以及维护体外系统的可用性并抑制这些基因的表达，为研究其转录调节。总体目标项目是为了分析顺式作用的转录调节元素角质和mimecan基因，以鉴定与之相互作用的角膜蛋白这些元素，并测试两个基因中的特定基因突变。这将解决以下问题：转录率如何在眼部与非眼组织中控制的米木和圆锥角膜？哪个主题角质和mimecan的转录监管区域的说明血清和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？这些功能是什么在同一组织中合成的多个mimecan mRNA转录本？是什么在多个多元中的顺式作用调节/定位元素的序列 Mimecan mRNA转录本和哪些蛋白质与这些元素结合？实验在体外进行的包括：DNase I超敏位点映射，荧光素酶记者基因测定，定点诱变/单个元素的缺失从监管区域确定其对功能的贡献，以及与结合的新转录因子的克隆和测序调节主题以表征其在驱动转录中的作用。给出克拉坦硫酸盐蛋白聚糖在提供角膜方面的重要性透明度，这里提出的研究可能会提供有关有关的新信息角膜特定的转录调节，因此提出了替代方案角膜KSPG在病理学中的治疗上调的靶标情况。此外，有关组织特异性转录的知识成功的体外生成组织需要调节移植。