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基因编码的kruppel样锌指蛋白含有5个串联锌指基序，与kruppel样蛋白Gli和Zic亚家族成员的同源性最高。此外，Glis1和-3的锌指结构域与果蝇的高同源性表明它可能是Glis1和-3的果蝇同源物。Northern blot分析显示Glis1和glis2 mRNA在成人肾脏中表达最丰富，而Glis3 mRNA在多个组织中表达。小鼠胚胎全坐骨原位杂交表明，Glis1-3在发育过程中以时间和空间方式表达。Glis1的表达在中胚层谱系的几个明确的结构中最为突出，包括颅面区域、鳃弓、体突、触须和毛囊、肢芽和肌切块，这表明Glis1在不同发育阶段发挥作用。Glis2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作用。Glis3在发育中的肾脏和睾丸的特定区域表达，并在神经发育期间以高度动态的模式表达。从E11.5到E12.5， Glis3在指间区强烈表达，注定会发生凋亡。在胚胎发育过程中观察到的Glis1-3表达的时空格局表明，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。共聚焦显微镜分析显示Glis1-3定位于细胞核。这些点状图案表明它们是一个更大的核蛋白复合体的一部分。Glis中的锌指区在这些蛋白的核定位中起着重要作用。电泳迁移率转移实验表明，Glis1-3能够结合含有GACCACCCAC一致序列的glis结合位点寡核苷酸。虽然单杂交分析显示，在几种细胞类型中Glis1-3不能诱导报告基因的转录，但缺失突变分析显示，Glis1-3具有很强的激活和抑制功能，表明这些蛋白可以作为转录的抑制和激活因子。Glis3被发现与GLi1相互作用，提示Glis和Gli信号通路之间存在相互作用。我们的研究结果表明，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，这些蛋白可能作用于超音hedgehog基因、Wnt、BMP或FGF信号通路的上游和/或下游。

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.