In this study, we describe the identification and characterization of a novel transcription factor GLI-similar 3 (GLIS3). GLIS3 is an 83.8 kDa nuclear protein containing five C2H2-type Krüppel-like zinc finger motifs that exhibit 93% identity with those of GLIS1, however, little homology exists outside their zinc finger domains. GLIS3 can function as a repressor and activator of transcription. Deletion mutant analysis determined that the N- and C-termini are required for optimal transcriptional activity. GLIS3 binds to the GLI-RE consensus sequence and is able to enhance GLI-RE-dependent transcription. GLIS3(DeltaC496), a dominant-negative mutant, inhibits transcriptional activation by GLIS3 and GLI1. Whole mount in situ hybridization on mouse embryos from stage E6.5 through E14.5 demonstrated that 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 GLIS3 expression observed during embryonic development suggests that it may play a critical role in the regulation of a variety of cellular processes during development. Both the repressor and activation functions of GLIS3 may be involved in this control.
The nuclear orphan receptor TAK1/TR4 functions as a positive as well as a negative regulator of transcription; however, little is known about the factors regulating or mediating its activity. Yeast two-hybrid analysis using the ligand-binding domain (LBD) of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27, which functions as a repressor of TAK1-mediated transactivation. TIP27 is a 27 kDa protein containing two zinc finger motifs. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. The region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1 while the TAK1-LBD from helix 3 until the C-terminus is required for the optimal interaction with TIP27. Pull-down assays demonstrated that the TIP27 physically interacts with TAK1 and supported the critical importance of the TID. Confocal microscopy showed that in the nucleus, TIP27 and TAK1 co-localize. TIP27 acts as a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve the inhibition of TAK1 homodimerization or DR1 binding but may be due to an effect on co-activator recruitment by TAK1. Our results indicate that TIP27 functions as a TAK1-selective repressor.
In this study, we describe the characterization of a gene encoding a novel Krü ppel-like protein, named Glis2. Glis2 encodes a relatively proline-rich, basic 55.8-kDa protein. Its five tandem Cys 2 -His 2 zinc finger motifs exhibit the highest homology to those of members of the Gli and Zic subfamilies of Krü ppel-like proteins. Confocal microscopic analysis demonstrated that Glis2 localizes to the nucleus. Analysis of the genomic structure of the Glis2 gene showed that it is composed of 6 exons separated by 5 introns spanning a genomic region of more than 7.5 kb. Fluorescence in situ hybridization mapped the mouse Glis2 gene to chromosome 16A3-B1. Northern blot analysis showed that the Glis2 gene encodes a 3.8-kb transcript that is most abundant in adult mouse kidney. By in situ hybridization, expression was localized to somites and neural tube, and during metanephric development predominantly to the ureteric bud, precursor of the collecting duct, and inductor of nephronic tubule formation. One-hybrid analysis using Glis2 deletion mutants identified a novel activation function (AF) at the N terminus. The activation of transcription through this AF domain was totally suppressed by two repressor functions just downstream from the AF. One of the repressor functions is contained within the first zinc finger motif. The level of transcriptional activation and repression varied with the cell line tested, which might be due to differences in cell typespecific expression of co-activators and co-repressors. Our results suggest that Glis2 behaves as a bifunctional transcriptional regulator. Both the activation and repressor functions may play an important role in the regulation of gene expression during embryonic development.
In this study, we describe the identification and characterization of a novel Krü ppel-like protein named Glisimilar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krü ppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; 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. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Glibinding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca 2؉ -dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development.
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