Mechanism Of Action And Functions Of The Gli-related Proteins Glis 1-3

Gli相关蛋白Glis 1-3的作用机制和功能

• 批准号: 8149074
• 负责人: Anton M Jetten
• 金额: $ 187.98万
• 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/8149074
• 关键词: Proteins

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.

GLIS1-3是最近在我们的实验室中发现的新基因。 GLIS1-3基因编码含有五个串联锌指基序的Kruppel样锌指蛋白，它们与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的锌指域与果蝇Gleeful/Lea Duck的同源性表现出很高的同源性，这表明它可能是Glis1和-3的果蝇同源物。北印迹分析表明，成年肾脏中GLIS1-3 mRNA的表达最丰富。小鼠胚胎上的整个原位杂交表明，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定义结构中最为突出，包括颅面区域，分支拱门，物种，振动和毛囊，肢体芽和肌肉群，表明在不同的发育阶段发挥作用。 GLIS2在肾脏和神经管中表达，表明在神经发生和肾脏发育中起作用。GLIS3在发育中的肾脏和睾丸以及在神经过程中具有高度动态的特定区域表达。从E11.5到E12.5 GLIS3在数字间区域强烈表达，这些区域被命中凋亡。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，它们可能在调节发育过程中的各种细胞过程中起关键作用。共聚焦显微镜分析表明，GLIS1-3位于核。穿刺模式表明它们是较大的核蛋白质复合物的一部分。 GLI中的锌指区域在这些蛋白质的核定位中起重要作用。电泳迁移率转移分析表明，GLIS1-3能够结合含有Gli结合位点共识序列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无法诱导记者的转录，但缺失突变体分析表明存在强激活和阻遏物功能，表明这些蛋白质可以充当转录的阻遏物和激活剂。发现GLIS3与GLI1相互作用，表明GLI和GLI信号通路之间的相互作用。我们的结果表明，GLIS1-3在胚胎发育的特定阶段的控制基因表达中可能起关键作用。我们的假设是，这些蛋白质可能在Sonic Hedgehog，Wnt，BMP或FGF信号传导途径的向上和/或下游作用。 为了了解GLIS2的生理功能，产生了缺乏GLIS2表达的小鼠。 Glis2突变体（Glis2Mut）小鼠最初看起来很健康，但由于进行性慢性肾脏疾病的发展而表现出比同窝型wt小鼠短得多的寿命。组织病理学分析显示，成年Glis2Mut小鼠的肾皮质发生了许多变化。这些包括管状萎缩和地下膜增厚，影响近端综合小管和肾小球。这伴随着单核（淋巴细胞）炎症细胞和间质/肾小球纤维化的浸润。纤维化，炎症性浸润以及肾小球和管状变化的严重程度随着年龄的增长而发展，与血液尿素氮和肌酐的增加相关，蛋白尿的发展以及水消耗量增加和尿液产量。最终，Glis2Mut小鼠过早地死于肾衰竭。通过微阵列分析比较了25和60天WT和GLIS2MUT小鼠的肾脏的基因表达谱图表明，在Glis2突变小鼠的肾脏中诱导了大量参与免疫反应/炎症和纤维化/组织重塑的基因，并包括几个细胞因子和外部外ellicallular Materix蛋白。我们的数据表明，GLIS2表达的缺乏会导致管状萎缩和进行性纤维化，最终导致肾衰竭。我们的研究表明，GLIS2在维持正常肾脏功能中起着至关重要的作用。 GLIS3：GLIS3在胰腺发育中起着至关重要的作用，并与新生儿糖尿病和甲状腺功能减退症（NDH）有关。我们检查了GLIS3转录调控机制的三个步骤：其转移到细胞核，DNA结合和转录活性。我们证明了不需要假定的双方核定位信号，但是第四个锌指的四面体构型对于GLIS3的核定位是必不可少的。我们将（g/c）tgggggt（A/C）识别为最佳，高亲和力GLIS3 DNA结合位点（GLIS-BS）的共有序列。所有五个锌指基序对于有效地结合GLIS3与Glis-BS都至关重要。我们表明GLIS3是（GLIS-BS）依赖性转录的有效诱导剂，并在其C末端包含反式激活函数。 NDH1患者观察到的GLIS3突变导致移码突变和C末端截短的GLIS3。我们证明，这种截断不会影响核定位，而是导致GLIS3反式激活活性的丧失。 GLIS3反式激活功能的损失可能导致NDH1中观察到的异常。 为了研究GLIS3的生理功能，产生了缺乏GLIS3表达的小鼠。我们的研究表明，GLIS3的功能障碍导致囊性肾脏疾病的发展，这表明GLIS3在维持正常肾功能中起着至关重要的作用。我们表明，Glis3定位于体内原发性纤毛，并在体外表明GLIS3是与纤毛相关的信号通路的一部分。此外，我们证明GLIS3与TAZ相互作用，TAZ本身与肾小球肾脏肾脏疾病有关。 TAZ可能是GLIS3介导的转录的共激活因子。此外，GLIS3 KO小鼠出生后3-5天患有新生儿糖尿病并死亡。发现GLIS3对于胰腺β细胞的产生至关重要。 GLIS3 KO小鼠中缺乏胰腺β细胞是导致糖尿病发展的。 另外，GLIS3可以调节胰岛素基因表达。