Transcriptional control of the aA-crystallin locus

描述（由申请人提供）：本申请的长期目标是阐明在α A-晶状体蛋白基因座的背景下调节α A-晶状体蛋白表达的转录控制机制。α A-晶状体蛋白是正常透镜透明度所必需的晶状体特异性蛋白质。人类α A-晶状体蛋白的突变导致白内障。转基因小鼠中α A-晶状体蛋白基因的靶向缺失同样引起透镜混浊。α A-晶状体蛋白的表达发生在透镜上皮细胞（透镜祖细胞）和透镜纤维细胞（终末分化细胞）中，并且由于其在分化的初级纤维中上调，因此其是透镜纤维细胞分化的优异标记物。本研究旨在确定和表征那些控制α A-晶状体蛋白在体内透镜上皮细胞和透镜纤维细胞中表达的调控区域。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功能性地描述小鼠aA-晶状体蛋白，（2）阐明aA-晶状体蛋白基因座的进化保守的远距离控制区（DCR）在体内的时间和空间功能，（3）定位与DCR相互作用的顺式调控位点和转录因子。这些目标将使用涉及转基因小鼠，时间和空间的基因表达模式的分析，使用一个乳糖Z标记，和DNA结合蛋白的生物化学表征与确定的DCR相互作用的综合方法来实现。转基因小鼠将使用转基因酶介导的盒交换（一种允许将单拷贝转基因插入特定染色体位点的方法）生产。所提出的研究的可行性得到了数据的支持，这些数据表明在小鼠和人α A-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在瞬时转染的透镜细胞中具有“增强子样”活性。这项工作的长期影响不仅是阐明α A-晶状体蛋白的转录调节，而且还收集数据，这些数据可用于合理设计用于透镜中转基因研究的工具，以探测正常的透镜过程，如透镜分化和维持，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.