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. 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. Glis proteins function as transcription factors and regulate the expression of target genes by binding Glis3 DNA-binding sites (Glis-BS) in their regulatory regions. The transcriptional activation is mediated by a transcriptional activation domain at the C-terminus of Glis1-3. To obtain insight into the physiological functions of Glis2, mice deficient in Glis1-3 were generated. Glis2 deficient mice appear initially healthy but exhibit a significantly shorter lifespan than littermate WT mice due to the development of progressive chronic kidney disease, nephronophthisis, that is accompanied by renal atrophy, fibrosis and inflammation. Ultimately Glis2 deficient mice die prematurely of renal failure. A large number of genes involved in immune responses/inflammation and fibrosis/tissue remodeling are induced in kidneys of Glis2 deficient mice. 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). 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. 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. We showed that Glis3 plays a critical role in the transcriptional regulation of insulin in pancreatic beta cells by recruiting CBP/p300, which may serve as a scaffold for the formation of a larger transcriptional regulatory complex containing Pdx1, NeuroD1, and MafA at the insulin promoter. Our data further suggest that the binding of Glis3 to the GlisBS and its recruitment of CBP/p300 are necessary to stabilize the interaction between Pdx1, NeuroD1, and MafA and their respective binding sites within the insulin promoter. This conclusion is consistent with findings showing that sustained expression of Glis3 is required for pancreatic cell generation in mice and humans, as well as for the maintenance of adult -cell functions, including insulin expression. Yeast two-hybrid analysis and mass spectrometry identified several Glis3-interacting proteins, including several HECT-ubiquitin ligases. We showed that Itch interacts with Glis3 and enhances Glis3 ubiquitination thereby reducing the stability of Glis3 protein and Glis3-mediated transcriptional activation. Glis3-deficient mice also develop glaucoma and congenital hypothyroidism, and exhibit an impaired spermatogenesis. Using Glis3-EGFP knocking mice, we were able to monitor Glis3 expression during embryonic development and postnatally. This study demonstrated that Glis3 is expressed in the thyroid follicles, early during spermatogenesis in gonocytes and spermatogonial progenitors, and in the pancreas in bipotent progenitors, endocrine progenitors, beta and PPY cells.
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