Mechanism Of Action And Functions Of Glis 1-3

Glis1-3是我们实验室最近发现的新基因。 Glis1-3 基因编码 Kruppel 样锌指蛋白，包含五个串联锌指基序，与 Kruppel 样蛋白的 Gli 和 Zic 亚家族成员表现出最高的同源性。此外，Glis1和-3的锌指结构域与果蝇gleeful/lame duck的锌指结构域表现出高度同源性，表明其可能是Glis1和-3的果蝇同源物。 Northern 印迹分析表明，Glis1 和 2 mRNA 的表达在成人肾脏中最为丰富，而 Glis3 在多种组织中表达。小鼠胚胎整体原位杂交表明 Glis1-3 在发育过程中以时间和空间方式表达。 Glis1 表达在中胚层谱系的几个明确结构中最为突出，包括颅面区域、鳃弓、体节、触毛和毛囊、肢芽和肌节，表明在不同发育阶段发挥作用。 Glis2 在肾脏和神经管中表达，表明在神经发生和肾脏发育中发挥作用。Glis3 在发育中的肾脏和睾丸的特定区域中表达，并且在神经形成过程中以高度动态的模式表达。从 E11.5 到 E12.5，Glis3 在指间区域强烈表达，这些区域注定会发生细胞凋亡。在胚胎发育过程中观察到的Glis1-3表达的时间和空间模式表明它们可能在发育过程中多种细胞过程的调节中发挥关键作用。共聚焦显微镜分析表明Glis1-3定位于细胞核。点状图案表明它们是更大的核蛋白复合物的一部分。 Glis 中的锌指区域在这些蛋白质的核定位中起着重要作用。电泳迁移率变动分析表明，Glis1-3 能够结合含有 Gli 结合位点共有序列 GACCACCCAC 的寡核苷酸。尽管单杂交分析表明，在几种细胞类型中，Glis1-3 无法诱导报告基因的转录，但缺失突变体分析揭示了强激活和阻遏功能的存在，表明这些蛋白可以充当转录阻遏蛋白和激活蛋白。 Glis3 被发现与 GLi1 相互作用，表明 Glis 和 Gli 信号通路之间存在相互作用。我们的结果表明，Glis1-3 可能在胚胎发育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可能在 sonic humighog、Wnt、BMP 或 FGF 信号通路的上游和/或下游发挥作用。 除了这些信号通路之间的联系之外，Glis 蛋白还与核受体 RORgamma 信号通路相互作用：Glis3 可以抑制 RORgamma 的转录激活。

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. In addition to links between these signaling pathways, Glis proteins interact with the nuclear receptor RORgamma signaling pathway: Glis3 can suppress the transcriptional activation by RORgamma.