In this study, we describe the cloning and sequence of a cDNA encoding a Krüppel-like zinc finger protein not reported previously, that was named GLI-similar 3 (GLIS3), based on its close relationship to GLIS1 (6
). GLIS3 contains a ZFD that exhibits highest identity, 93 and 81%, respectively, with the ZFD of GLIS1 (6
) and the apparent Drosophila
homolog gfl/lmd (11
). Interestingly, the exon–intron junctions within the region encoding the ZFD of GLIS3 are conserved with those of GLIS1 but distinct from the splice junctions in GLIS2, GLI and ZIC. This conservation in splice junctions indicates that GLIS1 and GLIS3 form a novel subfamily. Phylogenetic analysis further supports the conclusion that GLIS1, GLIS3 and gfl/lmd belong to a distinct subfamily of Krüppel-like zinc finger proteins different from, but closely related to, GLI, ZIC and GLIS2. GLIS1 and GLIS3 have little homology outside their ZFDs.
Analysis of its subcellular distribution showed that in several cell types GLIS3 localized predominantly to the nucleus. Deletion of a region containing the fifth zinc finger motif as well as a putative nuclear localization signal targeted the protein to the cytoplasm, suggesting that this region is required for the nuclear localization of GLIS3. In a number of zinc finger proteins, the zinc finger motifs have been implicated in nuclear localization (49
). Although the precise mechanism by which this region mediates nuclear transport is not yet quite understood, it likely involves protein–protein interaction. Whether the subcellular localization of GLIS proteins is regulated by protein–protein interactions, kinases or proteolytic processing, as has been reported for GLI proteins, has yet to be established (15
GLI and ZIC proteins regulate transcription by interacting with specific DNA response elements (GLI-REs) containing the consensus GACCACCCA in the promoter region of target genes (47
). GLIS3 is also able to interact with GLI-REs (Fig. ), as is GLIS1 (6
). However, GLI, ZIC and GLIS proteins can exhibit different affinities for distinct GLI-RE-like DNA elements. It has been shown that the sequences adjacent to the GACCACCCA consensus is important in determining the binding affinity of these zinc finger proteins to GLI-REs (52
). The third to fifth zinc finger motifs, which constitute the most highly conserved region within the ZFD of members of the GLI, ZIC and GLIS subfamilies (Fig. A), appear critical in the recognition of this specific sequence (47
). Differences in this region between these proteins may be responsible for the observed differences in their affinity for different GLI-REs.
Experiments examining the transcriptional activity of GLIS3 showed that it can function as a repressor and activator of transcription depending on the cell type analyzed. In CV-1 cells, GLIS3 repressed transcription in a dose-dependent manner, suggesting that in these cells GLIS3 acts as a repressor. However, in CHO cells GLIS3 functions as a weak transcriptional activator. The weak transactivation may be due to suboptimal interaction with the GLI-RE used, post-translational modifications or lack of appropriate co-activators. Deletion analysis demonstrated that deletion of the N-terminus up to His254 enhanced transactivation, while further deletion up to Tyr306 almost totally abolished GLIS3 activity. Similarly, deletion of its C-terminus up to Ser709 greatly diminished transcriptional activity. These results suggest that the N- and C-termini contain domains required for optimal transcriptional activation. In CHO cells, GLIS3 also induces GLI-RE-dependent transcription. The mutant GLIS3(ΔC496), lacking the activation function at the C-terminus, repressed the induction of transcription by either GLIS3 or GLI1, suggesting that this mutant acts as a dominant-negative factor. This inhibition may at least in part be due to competition for GLI-RE binding.
Repression and activation of transcription by GLI/ZIC/GLIS proteins is likely mediated through recruitment of intermediary proteins that function either as co-repressors or co-activators, respectively. These interactions involve specific motifs/domains in GLI/ZIC/GLIS. For example, the repression by a large number of Krüppel-like zinc finger proteins involves the specific repressor domain Krüppel-associated box (KRAB) (54
) or SCAN box (56
). The repressor function of the KRAB is mediated through its interaction with the co-repressor TIF1 (57
). However, the repressor activity of GLIS3 does not involve either a KRAB or SCAN box since the N-terminus of GLIS3 does not have any resemblance to these motifs. Studies are in progress to identify co-repressors and co-activators that mediate the activity of GLIS proteins.
We demonstrate that GLIS3 expression is restricted to specific tissues and structures in the mouse embryo. GLIS3 expression was first detected in the node, which is the murine equivalent of Spemann’s organizer in the frog and Hensen’s node in the chick (60
). However, GLIS3 is quickly down-regulated, as it is not expressed in the notochord, the midline structure derived from cells in the node. During neurulation, GLIS3 is first expressed in the early neural plate in the prospective hindbrain region and is then expressed in the mid/hindbrain junction and anterior forebrain by E8.5 and in the roof plate (the dorsal-most region of the neural tube) by E9.0. These expression domains sharpen over the next 3 days of development, but by E12.5 GLIS3 is mostly found in specific structures of the telencephalon. This highly dynamic pattern of GLIS3 expression during neurulation includes eye development. GLIS3 is first expressed in the dorsal optic vesicle and lens placode in the surface ectoderm. As eye development progresses, expression is evident in the lens, neural retina and nasal optic stalk. GLIS3 is prominently expressed in the mesenchyme of two outgrowths: the genital tubercle and limbs. Finally, during organogenesis GLIS3 is expressed in cell-specific patterns in lungs, trachea, testes and kidneys.
Expression of GLIS3 in multiple embryonic domains implicates this factor in a variety of cellular processes during development. Thus, GLIS3 may play a role in cell migration as it is expressed in structures that are sources of migratory cells: the node, the dorsal neural tube and anterior rhombic lip of the isthmus. However, GLIS3 is not expressed in the cells that migrate from these tissues (notochord, neural crest and granule cell precursors, respectively). In addition to the neural crest, the neural roof plate is also an organizing center that patterns the neural tube and functions as a source of progenitor cells that will give rise to the dorsal sensory interneurons. GLIS3 may play a role in either of these processes. Another role for GLIS3 is suggested by its strong expression in the interdigital regions before (E11.5) and during the onset (E12.5) of apoptosis. Thus, GLIS3 may play a role in the programmed cell death that removes this tissue and sculpts the digits.
One commonality in nearly all of the GLIS3 expression domains is that these are regions where signaling through BMPs or other members of the transforming growth factor (TGF)-β superfamily play a central role in embryogenesis. For example, regulation of BMP4 signaling is required for normal node function (61
). BMP signaling from the roof plate opposes SHH signaling from the floor plate and together these inductive signals generate dorsal/ventral positional patterning of the neural tube (62
). BMP4 is expressed in both the optic vesicle and surface ectoderm and is required for the inductive interactions between these tissues for normal eye development (63
). During limb development, BMPs and TGF-β2/3 are thought to be major determinants of interdigit cell death (64
), and Gdf5/6/7 are required in interphalangeal joints of the digits zone for proper joint development (66
). Gene inactivation studies in mouse embryos have shown that BMP7, which is expressed initially in the metanephric ureteric bud, is required for normal kidney development (67
), and TGF-β2/3 are required in lung development (69
). Finally, conditional Cre-mediated inactivation of BMP receptor 1A in facial mesenchyme has demonstrated that BMP signals are required for normal facial development (M. Lewandoski and T. Williams, unpublished observations). Therefore, we speculate that ligands of the TGF-β superfamily may be GLIS3 targets, however, we have not ruled out the possibility that GLIS3 may be downstream of BMP signaling.
As discussed above, gfl/lmd is the putative Drosophila
homolog of GLIS1 and GLIS3. gfl/lmd has been reported to play an important role in muscle differentiation. Embryos lacking lmd function show a loss of expression of Mef2 and stick-and-stones, two key differentiation and fusion genes, and are completely devoid of multinucleated muscle fibers (11
). Evidence has been provided that lmd may directly control transcription of the Mef2 gene. Although GLIS3 has been found in adult skeletal muscle, future studies have to identify its precise role in this tissue.
In the present study, we describe the identification of GLIS3 and provide evidence indicating that GLIS1 and GLIS3 form a distinct subfamily of Krüppel-like zinc finger proteins that is different from but closely related to GLI and ZIC. Whole mount in situ hybridization has demonstrated that during embryonic development GLIS3 is expressed in a temporal and spatial pattern suggesting that it may play a critical role in the regulation of a variety of cellular processes during development.