Transcriptional control of the Alpha A-crystallin locus

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aA-crystallin表达的转录控制机制。晶体蛋白是一种晶状体特异性蛋白，对晶状体的正常透明度至关重要。人α -晶状体蛋白突变导致白内障。在转基因小鼠中靶向缺失aa -晶体蛋白基因同样会导致晶状体混浊。aA-crystallin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）中均有表达，由于其在分化的原代纤维中表达上调，因此是晶状体纤维细胞分化的良好标志。本研究旨在鉴定和表征体内晶状体上皮细胞和晶状体纤维细胞中aa -晶体蛋白表达的调控区域。为了实现这一长期目标，我们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-crystallin进行功能描述；(2)阐明体内aA-crystallin基因座进化保守远控区（DCRs）的时空功能；(3)绘制与DCRs相互作用的顺式调控位点和转录因子。这些目标将通过一种综合的方法来实现，包括转基因小鼠，使用lac Z标记分析时间和空间基因表达模式，以及与鉴定的dcr相互作用的dna结合蛋白的生化表征。转基因小鼠将使用重组酶介导的盒式交换（Recombinase Mediated Cassette Exchange）产生，这种方法允许将转基因的单个拷贝插入特定的染色体位点。该研究的可行性得到了一些数据的支持，这些数据表明，在小鼠和人类的aa -晶体蛋白位点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。本工作的长期影响不仅在于阐明了aA-crystallin的转录调控，还在于收集了数据，可用于合理设计用于晶状体转基因研究的工具，以探索晶状体的正常过程，如晶状体的分化和维持，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.