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: 7968157
• 负责人: Anton M Jetten
• 金额: $ 118.91万
• 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/7968157
• 关键词: Adhesions; Adult; Affect; Affinity; Age; Apoptosis; Atrophic; Basement membrane; Binding; Binding Sites; Blood Urea Nitrogen; Branchial arch structure; C-terminal; Cell Nucleus; Cell physiology; Chronic Kidney Failure; Cilia; Consensus Sequence; Creatinine; Cystic Kidney Diseases; DNA Binding; Development; Diabetes Mellitus; Drosophila genus; Ducks; Electrophoretic Mobility Shift Assay; Embryo; Embryonic Development; Erinaceidae; Exhibits; Extracellular Matrix Proteins; Fibroblast Growth Factor; Fibrosis; Frameshift Mutation; Functional disorder; Gene Expression; Genes; Genetic Transcription; Hair follicle structure; Homologous Gene; Human; Hypothyroidism; Immune response; In Situ Hybridization; In Vitro; Infiltration; Inflammation; Inflammatory; Inflammatory Infiltrate; Kidney; Kidney Diseases; Kidney Failure; Kruppel protein; Laboratories; Limb Bud; Longevity; Lymphocyte; Maintenance; Malignant Neoplasms; Mediating; Microarray Analysis; Microscopic; Molecular Profiling; Mononuclear; Mus; Mutant Strains Mice; Mutation; Neural tube; Northern Blotting; Nuclear; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Oligonucleotides; Osteogenesis; Output; Pancreas; Patients; Pattern; Physiological; Play; Polycystic Kidney Diseases; Proteins; Proteinuria; Regulation; Renal function; Reporter; Role; Severities; Signal Pathway; Somites; Staging; Structure; Syndrome; Testis; Tissues; Transactivation; Transcription Coactivator; Transcriptional Regulation; Tubular formation; Urine; Water consumption; Zinc Fingers; cell type; craniofacial; cytokine; human disease; in vivo; insight; interstitial cell; kidney cortex; member; mutant; neonatal diabetes mellitus; neurogenesis; novel; protein complex; 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 Glis1-3 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.

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，Glis1-3 mRNA 的表达在成人肾脏中最为丰富。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、触须和毛囊、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，表明这些蛋白质可以充当转录的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。 为了深入了解 Glis2 的生理功能，产生了 Glis2 表达缺陷的小鼠。 Glis2 突变 (Glis2mut) 小鼠最初看起来很健康，但由于发展为进行性慢性肾病，其寿命明显短于同窝 WT 小鼠。组织病理学分析揭示了成年 Glis2mut 小鼠肾皮质的许多变化。这些包括影响近曲小管和肾小球的肾小管萎缩和基底膜增厚。这伴随着单核（淋巴细胞）炎症细胞的浸润和间质/肾小球纤维化。纤维化、炎症浸润以及肾小球和肾小管变化的严重程度随着年龄的增长而进展，并与血尿素氮和肌酐的增加、蛋白尿的发生以及水​​消耗和尿量的增加相关。最终 Glis2mut 小鼠因肾衰竭过早死亡。通过微阵列分析比较25日龄和60日龄WT小鼠和Glis2mut小鼠肾脏的基因表达谱，发现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，包括多种细胞因子、粘附和细胞外基质蛋白。我们的数据表明，Glis2 表达缺陷会导致肾小管萎缩和进行性纤维化，最终导致肾衰竭。我们的研究表明，Glis2 在维持正常肾功能中发挥着关键作用。 Glis3：Glis3 在胰腺发育中发挥着关键作用，并与新生儿糖尿病和甲状腺功能减退症 (NDH) 综合征有关。我们研究了 Glis3 转录调控机制中关键的三个步骤：其转位至细胞核、DNA 结合和转录活性。我们证明，假定的二分核定位信号不是必需的，但第四个锌指的四面体构型对于 Glis3 的核定位至关重要。我们将 (G/C)TGGGGGGT(A/C) 确定为最佳高亲和力 Glis3 DNA 结合位点 (Glis-BS) 的共有序列。所有五个锌指基序对于 Glis3 与 Glis-BS 的有效结合都至关重要。我们表明，Glis3 作为 (Glis-BS) 依赖性转录的有效诱导剂发挥作用，并在其 C 末端包含反式激活功能。在 NDH1 患者中观察到的 Glis3 突变导致移码突变和 C 端截短的 Glis3。我们证明这种截短不会影响核定位，但会导致 Glis3 反式激活活性丧失。 Glis3 反式激活功能的丧失可能是 NDH1 中观察到的异常的原因。 为了研究 Glis3 的生理功能，产生了 Glis3 表达缺陷的小鼠。我们的研究表明，Glis3 功能障碍会导致囊性肾病的发生，表明 Glis3 在维持正常肾功能中发挥着关键作用。我们发现 Glis3 在体内和体外定位于初级纤毛，表明 Glis3 是纤毛相关信号通路的一部分。此外，我们证明 Glis3 与 TAZ 相互作用，而 TAZ 本身与肾小球囊性肾病有关。 TAZ 可能作为 Glis3 介导的转录的共激活剂发挥作用。