Transcriptional control of the Alpha A-crystallin locus

描述(申请人提供)：本申请的长期目标是阐明调控AA-晶体蛋白在其基因座上表达的转录调控机制。AA-晶状体蛋白是一种晶状体特有的蛋白质，对正常晶状体的透明度至关重要。人类AA-晶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。AA-晶体蛋白在晶状体上皮细胞(晶状体前体细胞)和晶状体纤维细胞(终末分化细胞)中均有表达，在分化中的初级纤维中表达上调，是晶状体纤维细胞分化的良好标志物。本研究试图在体内识别和表征在晶状体上皮细胞和晶状体纤维细胞中控制AA-晶体蛋白表达的调控区域。为了实现这一长期目标，提出了以下具体目标：(1)利用标准化和随机整合位点在转基因小鼠模型中对小鼠AA-晶体蛋白进行功能描述；(2)阐明AA-晶体蛋白基因座进化保守的远程控制区(DCRS)在体内的时空功能；(3)定位与DCRS相互作用的顺式调控位点和转录因子。这些目标将通过使用转基因小鼠的综合方法、使用lac Z标记分析时空基因表达模式以及与已识别的DCRS相互作用的DNA结合蛋白的生化特征来实现。转基因小鼠将使用重组酶介导盒交换产生，这是一种允许将转基因的单一副本插入特定染色体位置的方法。在小鼠和人类AA晶体蛋白基因座中存在进化保守的非编码假定DCRS的数据支持了所提出的研究的可行性。这些DCRs在瞬时转基因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-晶体蛋白的转录调控，而且还收集了数据，可用于合理设计用于晶状体转基因研究的工具，以探测晶状体分化和维护等正常晶状体过程，并诱导异常过程，如特定的白内障模型。

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.