Mechanism Of Action And Functions Of The Gli-related Proteins 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Glis1-3 mrna在成人肾脏中表达最丰富。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 Glis1-3 mRNAs are most abundant in adult kidney. 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 obtain insight into the physiological functions of Glis2, mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2mut) mice appear initially healthy but exhibit a significantly shorter lifespan than littermate WT mice due to the development of progressive chronic kidney disease. Histopathological analysis revealed a number of changes in the renal cortex of adult Glis2mut mice. These included tubular atrophy and basement membrane thickening affecting the proximal convoluted tubules and glomeruli. This was accompanied by infiltration of mononuclear (lymphocytic) inflammatory cells and interstitial/glomerular fibrosis. The severity of the fibrosis, inflammatory infiltrates, and the glomerular and tubular changes progressed with age and correlated with increases in blood urea nitrogen and creatinine, the development of proteinuria and increased water consumption and urine output. Ultimately Glis2mut mice die prematurely of renal failure. Comparison of the gene expression profiles of kidneys from 25 and 60 day old WT and Glis2mut mice by microarray analysis showed that a large number of genes involved in immune responses/inflammation and fibrosis/tissue remodeling are induced in kidneys of Glis2 mutant mice, and included several cytokines, adhesion and extracellular matrix proteins. Our datademonstrate that deficiency in Glis2 expression leads to tubular atrophy and progressive fibrosis that ultimately results in renal failure. Our study indicates that Glis2 plays a critical role in the maintenance of normal kidney functions. Glis3: Glis3 plays a critical role in pancreatic development and has been implicated in a syndrome with neonatal diabetes and hypothyroidism (NDH). We examined three steps critical in the mechanism of the transcriptional regulation by Glis3: its translocation to the nucleus, DNA binding, and transcriptional activity. We demonstrate that the putative bipartite nuclear localization signal is not required, but the tetrahedral configuration of the fourth zinc finger is essential for the nuclear localization of Glis3. We identify (G/C)TGGGGGGT(A/C) as the consensus sequence of the optimal, high affinity Glis3 DNA-binding site (Glis-BS). All five zinc finger motifs are critical for efficient binding of Glis3 to Glis-BS. We show that Glis3 functions as a potent inducer of (Glis-BS)-dependent transcription and contains a transactivation function at its C-terminus. A mutation in Glis3 observed in NDH1 patients results in a frameshift mutation and a C-terminal truncated Glis3. We demonstrate that this truncation does not effect the nuclear localization but results in the loss of Glis3 transactivating activity. The loss in Glis3 transactivating function may be responsible for the abnormalities observed in NDH1. To study the physiological function of Glis3, mice deficient in the expression of Glis3 were generated. Our study demonstrates that dysfunction of Glis3 leads to development of cystic renal disease suggesting that Glis3 plays a critical role in maintaining normal renal functions. We show that Glis3 localizes to the primary cilium in vivo and in vitro suggesting that Glis3 is part of a cilium-associated signaling pathway. In addition, we demonstrate that Glis3 interacts with the TAZ, which itself has been implicated in glomerulocystic kidney disease. TAZ may function as a co-activator of Glis3-mediated transcription. In addition, Glis3 KO mice develop neonatal diabetes and die 3-5 days after birth. Glis3 was found to be essential for pancreatic beta cells generation. The lack of pancreatic beta cells in Glis3 KO mice is responsible for the development of diabetes. In addition, Glis3 can regulate insulin gene expression.