Transcriptional control of the aA-crystallin locus

aA-晶状体蛋白位点的转录控制

• 批准号: 6724796
• 负责人: Ales Cvekl
• 金额: $ 37.58万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724796
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; cataract; cell differentiation; crystallins; epithelium; fiber cell; gel mobility shift assay; gene expression; genetic enhancer element; genetic mapping; genetic promoter element; genetic regulatory element; genetically modified animals; laboratory mouse; lens; molecular cloning; protein structure function; site directed mutagenesis; stem cells; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): The long-term goal of this application is to elucidate the trancriptional control mechanisms that regulate the expression of aA-crystallin in the context of its locus. aA-crystallin is a lens-specific protein essential for normal lens transparency. Mutations in human aA-crystallin cause cataracts. Targeted deletion of the aA-crystallin gene in transgenic mouse likewise causes lens opacification. Expression of aA-crystallin occurs both in the lens epithelium (lens progenitor cells) and in lens fiber cells (terminally differentiated cells) and since it is up-regulated in the differentiating primary fibers it is an excellent marker for lens fiber cell differentiation. This study seeks to identify and characterize those regulatory regions controlling aA-crystallin expression in the lens epithelium and the lens fiber cells in vivo. In order to carry out this long-term goal the following specific aims are proposed: (1) To functionally delineate the mouse aA-crystallin in a transgenic mouse model using standardized and random integration sites, (2) To elucidate the temporal and spatial functions of evolutionary conserved distant control regions (DCRs) of the aA-crystallin locus in vivo, and (3) To map cis-regulatory sites and transcription factors interacting with the DCRs. These aims will be achieved using an integrative approach involving transgenic mice, analyses of temporal and spatial gene expression patterns using a lac Z marker, and biochemical characterization of DNA-binding proteins interacting with the identified DCRs. Transgenic mice will be produced using the Recombinase Mediated Cassette Exchange, a method allowing insertion of a single copy of the transgene into a specific chromosomal site. The feasibility of the proposed study is supported by data demonstrating the presence of evolutionary conserved non-coding putative DCRs in the mouse and human aA-crystallin loci. These DCRs harbor "enhancer-like" activities in transiently transfected lens cells. The long-range impact of this work is not only the elucidation of transcriptional regulation of aA-crystallin, but also collection of data that can be used for a rational design of tools used for transgenic studies in the lens to probe normal lens processes such as lens differentiation and maintenance, and to induce abnormal processes such as specific cataract models.

描述（由申请人提供）：本申请的长期目标是阐明在其基因座背景下调节αA-晶状体蛋白表达的转录控制机制。 aA-晶状体蛋白是一种晶状体特异性蛋白质，对于正常晶状体透明度至关重要。人类αA-晶状体蛋白的突变会导致白内障。转基因小鼠中 aA-晶状体蛋白基因的定向缺失同样会导致晶状体混浊。 αA-晶状体蛋白的表达发生在晶状体上皮（晶状体祖细胞）和晶状体纤维细胞（终末分化细胞）中，并且由于它在分化的初级纤维中上调，因此它是晶状体纤维细胞分化的极好标志物。本研究旨在鉴定和表征控制体内晶状体上皮和晶状体纤维细胞中αA-晶状体蛋白表达的调节区域。为了实现这一长期目标，提出了以下具体目标：（1）使用标准化和随机整合位点在转基因小鼠模型中功能性地描述小鼠aA-晶状体蛋白，（2）阐明时间和空间功能体内aA-晶状体蛋白基因座的进化保守的远程控制区（DCR）的分析，以及（3）绘制与DCR相互作用的顺式调节位点和转录因子。这些目标将通过使用涉及转基因小鼠的综合方法、使用 lac Z 标记分析时间和空间基因表达模式以及与已识别的 DCR 相互作用的 DNA 结合蛋白的生化表征来实现。转基因小鼠将使用重组酶介导的盒交换来生产，该方法允许将转基因的单个拷贝插入特定的染色体位点。所提出研究的可行性得到了数据的支持，数据证明了小鼠和人类 aA-晶状体蛋白基因座中存在进化保守的非编码推定 DCR。这些 DCR 在瞬时转染的晶状体细胞中具有“增强子样”活性。这项工作的长期影响不仅是阐明aA-晶状体蛋白的转录调控，而且还收集了可用于合理设计用于晶状体转基因研究的工具的数据，以探测正常的晶状体过程，例如晶状体分化和维护，并诱导异常过程，例如特定的白内障模型。