Mechanism Of Action And Functions Of The Gli-related Pro

Gli相关Pro的作用机制和功能

• 批准号: 7170025
• 负责人: Anton M Jetten
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7170025
• 关键词: biological signal transduction; confocal scanning microscopy; developmental genetics; embryogenesis; gel mobility shift assay; gene expression; genetically modified animals; human tissue; in situ hybridization; laboratory mouse; nephrogenesis; neurogenesis; northern blottings; nuclear transport; protein localization; protein protein interaction; protein structure function; protein transport; transcription factor

Glis1-3 are novel genes recently identified in our laboratory. The Glis1-3 genes encode Kruppel-like zinc finger proteins containing five tandem zinc finger motifs that exhibit highest homology with those of members of the Gli and Zic subfamilies of Kruppel-like proteins. In addition, the zinc finger domain of Glis1 and -3 exhibit high homology with that of Drosophila gleeful/lame duck suggesting that it may be the Drosophila homologue of Glis1 and -3. Northern blot analysis showed that expression of the Glis1 and 2 mRNA is most abundant in adult kidney while Glis3 is expressed in several tissues. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1-3 are expressed in a temporal and spatial manner during development. Glis1 expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes suggesting a role at different stages of development. Glis2 was expressed in kidney and neural tube suggesting a role in neurogenesis and kidney development.Glis3 is expressed in specific regions in developing kidney and testis and in a highly dynamic pattern during neurulation. From E11.5 through E12.5 Glis3 was strongly expressed in the interdigital regions, which are fated to undergo apoptosis. The temporal and spatial pattern of Glis1-3 expression observed during embryonic development suggests that they may play a critical role in the regulation of a variety of cellular processes during development.Confocal microscopic analysis showed that Glis1-3 are localized to the nucleus. The punctated pattern suggests that they are part of a larger nuclear protein complex. The zinc finger region in Glis plays an important role in the nuclear localization of these proteins. Electrophoretic mobility shift assays demonstrated that Glis1-3 are able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1-3 are unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation and repressor functions suggesting that these proteins can function as repressors and activators of transcription. Glis3 was found to interact with GLi1 suggesting interaction between the Glis and Gli signaling pathways. Our results suggest that Glis1-3 may play a critical role in the control of gene expression during specific stages of embryonic development. Our hypothesis is that these proteins may act up- and/or downstream of sonic hedgehog, Wnt, BMP, or FGF signaling pathways. To study the physiological function of Glis2 and Glis3 mice deficient in the expression of these proteinswere generated. Glis2-Ko mice exhibit a kidney phenotype: mice die early from renal failure. Glis1-Ko mice die shortly after birth. The cause of these phenotypes are being studied.

Glis 1 -3是本实验室最近发现的新基因。Glis 1 -3基因编码Kruppel样锌指蛋白，其含有5个串联锌指基序，与Kruppel样蛋白的Gli和Zic亚家族的成员具有最高的同源性。此外，Glis 1和Glis 3的锌指结构域与果蝇的锌指结构域有很高的同源性，可能是Glis 1和Glis 3在果蝇中的同源物。北方印迹分析显示Glis 1和2 mRNA在成人肾脏中表达最丰富，而Glis 3在多种组织中表达。小鼠胚胎原位杂交结果表明，Glis 1 -3在胚胎发育过程中具有时空表达。Glis 1的表达是最突出的几个定义的结构中胚层谱系，包括颅面区，鳃弓，体节，触须和毛囊，肢芽，和肌节，这表明在不同的发展阶段的作用。Glis 2在肾脏和神经管中表达，提示其在神经发生和肾脏发育中起重要作用; Glis 3在发育中的肾脏和睾丸的特定区域表达，并在神经形成过程中呈高度动态表达。从E11.5到E12.5，Glis 3在趾间区强烈表达，这是注定要经历凋亡的。Glis 1 -3在胚胎发育过程中表达的时间和空间模式表明，它们可能在发育过程中的各种细胞过程的调节中发挥关键作用。点状图案表明它们是更大的核蛋白复合物的一部分。Glis中的锌指区在这些蛋白质的核定位中起重要作用。电泳迁移率变动分析表明，Glis 1 -3能够结合寡核苷酸含有Gli-binding位点的共识序列GACCACCCAC。虽然单杂交分析表明，在几种细胞类型的Glis 1 -3是无法诱导转录的报告，缺失突变体的分析显示存在一个强大的激活和阻遏功能，这表明这些蛋白质可以作为阻遏物和激活转录。发现Glis 3与GLi 1相互作用，表明Glis和Gli信号通路之间存在相互作用。我们的研究结果表明，Glis 1 -3可能在胚胎发育的特定阶段的基因表达控制中发挥关键作用。我们的假设是，这些蛋白质可能作用于音刺猬，Wnt，BMP或FGF信号通路的上游和/或下游。 为了研究Glis 2和Glis 3的生理功能，产生了这些蛋白表达缺陷的小鼠。Glis 2-Ko小鼠表现出肾脏表型：小鼠死于肾衰竭。Glis 1-Ko小鼠出生后不久死亡。这些表型的原因正在研究中。