Identification of Nuclear Localization, DNA Binding, and Transactivating Mechanisms of Krüppel-like Zinc Finger Protein Gli-Similar 2 (Glis2)

Abstract

Gli-similar 1-3 (Glis1-3) constitute a subfamily of Krüppel-like zinc finger (ZF) transcription factors that are closely related to the Gli protein family. Mutations in GLIS2 are linked to nephronophthisis, a chronic kidney disease characterized by renal fibrosis and atrophy in children and young adults. Currently, very little information exists about the mechanism of action of Glis2, its target genes, or the signaling pathways that regulate its activity. In this study, we show that a region within ZF3 is required for the nuclear localization of Glis2. Analysis of Glis2 DNA binding demonstrated that Glis2 binds effectively to the consensus Glis binding sequence (GlisBS) (G/C)TGGGGGGT(A/C). Although Glis2 was unable to induce transactivation of a GlisBS-dependent reporter, it effectively inhibited the GlisBS-mediated transactivation by Gli1. Mutations that disrupt the tetrahedral configuration of each ZF within Glis2 abolished Glis2 binding to GlisBS and also abrogated its inhibition of Gli1-mediated transactivation. In contrast, Glis2 was able to activate the murine insulin-2 (Ins2) promoter by binding directly to two GlisBS elements located at ؊263 and ؊99 within the Ins2 promoter. Phosphomimetic mutation of Ser 245 inhibited the binding of Glis2 to GlisBS and dramatically affected its transactivation of the Ins2 promoter and its ability to inhibit GlisBS-dependent transactivation by Gli1. In this study, we demonstrate that Glis2 can function as a transcriptional activator and that post-translational modification within its DNA-binding domain can regulate its transcriptional activity. This control may play a critical role in the Glis2-dependent regulation of target genes and renal function.

Interaction with the Bardet-Biedl Gene Product TRIM32/BBS11 Modifies the Half-life and Localization of Glis2/NPHP7

“…We speculated that the TRIM32-mediated shift in Glis2 K48/ K63 polyubiquitylation may also alter the interaction partners of Glis2, affecting its subcellular localization. Immunofluorescence experiments in HEK 293T cells using YFP-tagged Glis2 (YFP.Glis2) revealed a diffuse nuclear localization of Glis2 consistent with previous observations (18) (Fig. 8A).…”

“…10A). Although Glis2 interacts with CtBP1 to function as a transcriptional repressor in most instances (12), Glis2 activates the mIns2 (mouse insulin-2) promoter by binding to the Glis binding sites of the mIns2 promoter (18). Here, TRIM32 had a modest repressor function, inhibiting the Glis2-mediated mIns2 activation by ϳ50% (Fig.…”

“…In the absence of NPHP7/Glis2, gene products implicated in epithelial to mesenchymal transition are upregulated, including TGF␤, Vimentin, Snail, and Slug. NPHP7/ Glis2 localizes primarily to the nucleus, requiring a region within zinc finger 3 (18), and can recruit other interacting proteins such as p120 catenin to the nucleus (19). However, both NPHP7/Glis2 and Glis3 have also been identified in the primary cilium (8,20).…”

Modulation of the Transactivation Function and Stability of Krüppel-like Zinc Finger Protein Gli-similar 3 (Glis3) by Suppressor of Fused

“…7B). Previously, we reported that Glis2 was able to activate the mIP(Ϫ696)-Luc reporter in HEK293 cells (21). We therefore examined whether SUFU had any effect on the transactivation of the Ins2 promoter by Glis2.…”

“…Generation of Reporter and Expression Constructs-The generation of p3xFlag-CMV-Glis1, p3xFlag-CMV-Glis2, p3xFlag-CMV-Glis3, p3xFlagCMV-Glis3⌬N302, p3xFlag-CMVGlis2⌬N58, p3xFlag-CMV-Glis2⌬N160, and p3xFlag-CMVGlis2⌬C169 was described previously (6,9,20,21). The plasmids p3xFlag-CMV-Glis3⌬N34, -65, -155, -288, -295, -319, -333, -355, and -388 and p3xFlag-CMV-Glis3⌬C389 were generated by PCR amplification of the respective fragments and inserting them into the EcoRI and BamHI restriction sites of the p3xFlag-CMV10 expression vector (Sigma).…”