Abstract
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.
Highlights
The Glis family of Krüppel-like zinc finger transcription factors, which is comprised of three members designated Glis1-3, contain a zinc finger domain (ZFD) consisting of five Cys2-His2 zinc finger motifs that exhibit high homology with the ZFDs of members of the Gli and Zic Krüppel-like zinc finger families [1]
We previously reported that the central region of Gli-similar 3 (Glis3) containing the ZFD and the C-terminal transactivation domain are essential for Glis3-mediated transcriptional activation [1,33]
We discover a new function for the N-terminus of Glis3 in the regulation of Glis3 stability and its transcriptional activity
Summary
The Glis family of Krüppel-like zinc finger transcription factors, which is comprised of three members designated Glis, contain a zinc finger domain (ZFD) consisting of five Cys2-His zinc finger motifs that exhibit high homology with the ZFDs of members of the Gli and Zic Krüppel-like zinc finger families [1]. Genetic aberrations within the GLIS3 locus are associated with a rare syndrome characterized by neonatal diabetes and hypothyroidism and may include polycystic kidney disease, hepatic fibrosis, glaucoma, and mild mental retardation depending on the nature of the mutation [8,9]. A similar phenotype, including neonatal diabetes, polycystic kidney disease, and hypothyroidism, is observed in mice lacking functional Glis3 [3,4,10]. Additional evidence suggests that Glis directly regulates insulin transcription in mature beta cells by binding two GlisBS located within the proximal promoter of the preproinsulin gene [3,5,18]. While the ZFD is involved in DNA binding, transcriptional activation of gene expression by Glis is mediated through a transactivation domain located within its C-terminus [1,18,19,20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
