Physical and Functional Interactions between Zic and Gli Proteins*

Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. We provide evidence that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.

Zic and Gli transcription factors share a highly conserved zinc finger domain and have critical roles in multiple developmental processes. In human, mutations in ZIC2, ZIC3, and GLI3 genes result in various developmental abnormalities. ZIC2 results in malformation of the forebrain (holoprosencephaly), ZIC3 in a disturbance of the left to right body axis (heterotaxy), and GLI3 in complex anomalies of the brain and digits (cephalopolysyndactyly syndrome) (1)(2)(3). Studies in other vertebrates indicated that Zic1, Zic2, Zic3, Gli1, Gli2, and Gli3 are involved in multiple aspects of the neural and skeletal development (4 -14). Zic and Gli families are also critical in invertebrate development as shown by the studies on their Drosophila homologues, Odd-paired (15) and Cubitus interruptus (Ci) (16).
Although a number of studies suggest the importance of the two zinc finger protein families, the relationship between them has not been fully understood. However, recent studies have shown significant Zic-Gli genetic interaction in neural and skeletal patterning. Xenopus Zic2 and Gli2 are counter-active in the patterning of neural tube along the dorsoventral axis (13). On the other hand, the double mutation of Zic1 and Gli3 showed a synergistic disturbance in the segmentation of the vertebral lamina (17).
Gli proteins bind a consensus nonamer target DNA sequence (GLI-BS) (18) to which Zic proteins can also bind (19). However, we recently found that the DNA-binding affinity of the Zic proteins was lower than that of Gli (20) and that Zic proteins significantly enhance gene expression but less efficiently in the absence of GLI-BS. When Zic and Gli are expressed together in cultured cells, they synergistically enhance, or mutually suppress, GLI-BS-mediated transcription depending on the cell type (20). Here we show that Zic and Gli proteins physically interact through their zinc finger domains and regulate each other's subcellular localization and transcriptional activity.
Immunoprecipitation and GST Pull-down Assays-293T cells were transiently cotransfected with appropriate expression constructs using Superfect or Effectene (Qiagen). After 48 h, cells were lysed in immunoprecipitation buffer (25 mM Hepes, pH 7.2, 0.5% Nonidet P-40, 150 mM NaCl, 50 mM NaF, 2 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin) at 4°C. Immunoprecipitation was performed using anti-HA Y-11 polyclonal antibody (Santa Cruz Biotechnology). Bound material was detected by immunoblotting with anti-Flag M2 monoclonal antiboby (Sigma). For GST pull-down assays, GST fusion proteins were incubated with bacterially expressed Flag-tagged GLI3-(461-639) or protein extracts from 293T cells transfected with different expression constructs in the immunoprecipitation buffer for 2 h at 4°C. Bound proteins were separated by SDS-polyacrylamide gel electrophoresis followed by immunoblotting using the anti-Flag antibody.
Subcellular Localization Studies-NIH3T3 cells and 293T cells were transiently transfected with appropriate expression constructs. 24 h after transfection, cells were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 20 min at room temperature and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline for 2 min. The cells were incubated in blocking buffer (1% bovine serum albumin and 0.1% Triton X-100 in phosphate-buffered saline) for 1 h at room temperature and then incubated with the anti-HA antibody and the anti-Flag antibody. The bound antibodies were detected by Alexa 488conjugated anti-rabbit IgG or Alexa 568-conjugated anti-mouse IgG antibodies (Molecular Probes, Inc.).
Luciferase Assay-Cells were cultured in 24-well dishes. NIH3T3 cells were transfected using Superfect with appropriate expression constructs. At 30 h after transfection, luciferase activities of the cells were measured as described (20). MNS70 cells (27,28) were transfected using FGENE6 (Roche Molecular Biochemicals) and assayed 48 h after transfection.

RESULTS AND DISCUSSION
Physical Interaction between Zic and Gli Proteins-To investigate the physical interaction between Zic and Gli, 293T cells were cotransfected with HA-tagged mouse Zic and Flag-tagged human or mouse Gli, and the resultant cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Flag antibody. Bands corresponding to GLI1, Gli2, and GLI3 were detected (Fig. 1A).
Translocation of Gli Proteins by Zic Proteins-Next we examined the subcellular localization of Zic and GLI proteins. Transfected HA-tagged Zic1, Zic2, and Zic3 were located in cell nuclei in all of the cell lines tested (NIH3T3, 293T, C3H10T1/2, COS7) (Fig. 2, A, B, I, and J, data not shown), whereas the subcellular localization of Flag-tagged GLI proteins has been found to vary in different contexts (8,29). In NIH3T3 and 293T cells, both GLI1 and GLI3 proteins were located predominantly in the cytoplasm (Fig. 2, C, D, H, K, L, and P). Coexpression of Zic1 resulted in GLI1 and GLI3 proteins being translocated to the nucleus in varying levels (Fig. 2, E and M). This tendency was clearest in the case of GLI3 in NIH3T3 cells and GLI1 in 293T cells (Fig. 2, H and P). A mutant GLI3 protein lacking residues 548 -624 (GLI3 ⌬ZF3-5) was not translocated by the coexpressed Zic1 (Fig. 2H). Enhancement of the nuclear translocation of GLI proteins was also observed with any combinations of Zic-/Gli proteins (data not shown) and in other cell lines (C3H10T1/2, HeLa, and COS7, data not shown).
Zic and Gli Proteins Regulate Each Other's Transcriptional Activity through the Zinc Finger Domains-Zic proteins activate transcription from the thymidine kinase (TK) promoter in a process that is partially dependent on GLI-BS (20, Fig. 3). However Gli proteins specifically require GLI-BS for transcriptional regulation and have essentially no effect on the promoter in the absence of GLI-BS (Ref. 20; Fig. 3). To clarify the significance of the Zic-Gli association in transcriptional regulation, we performed Zic-Gli cotransfection experiments using TK promoter-luciferase reporter constructs with and without GLI-BS (pGBS-TK-luc and pTK-luc, respectively) in NIH3T3 cells. When GLI1 and Zic1 (Fig. 3A) or GLI1 and Zic2 (Fig. 3B) were cotransfected, reporter gene expression was synergistically activated both in the presence and absence of GLI-BS. This synergistic activation was also observed in a Shh-responsive cell line (MNS70) (Fig. 3C). The level of synergistic increase was not influenced by the presence of an Shh signal (Fig. 3C), suggesting that the Shh signal does not regulate the Zic-Gli interaction. By contrast, full-length GLI3 enhanced reporter gene expression when coexpressed with a general transcription cofactor, CBP (Ref. 23; Fig. 3D). When Zic1 was coexpressed with GLI3 and CBP, a marked increase was observed in comparison with GLI3 and CBP coexpression (Fig. 3D). Conversely, when Zic1 was coexpressed with a carboxyl-terminally truncated GLI3 protein (Ref. 30; GLI3 18 -829 in Fig. 3D), the Zic1-mediated reporter gene activation was suppressed both in the pGBS-TK-luc and pTK-luc reporters (Fig. 3D). GLI3 ⌬ZF3-5 and a carboxyl-terminally truncated construct lacking ZF3-5 (GLI3-(1-547)) had no effect on reporter gene expression (Fig. 3D), suggesting an essential role for the ZF3-5 domain in the transcriptional interaction between Zic1 and GLI3. These findings therefore indicate that the Zic proteins enable the GLI proteins to participate in transcriptional regulation through a protein-to-protein association. The numbers refer to amino acids. The total amount of DNA was adjusted to 500 ng with control vector pCMVtag2. The reporter plasmid with (pGBS-TK-luc, black) and without (pTK-luc, white) GLI-BS was included in each transfection experiment. The luciferase gene without the TK promoter was not activated by either Gli or Zic. The error bars represent standard deviations. Luciferase activities are indicated as values relative to those of cells transfected with reporter plasmids and empty expression vectors.
The Zic-Gli associations may be involved in transcriptional regulation as well as in synergistic or antagonistic effects in different developmental contexts (13,17). Synergistic activation can be explained in part by enhancement of the nuclear localization of Gli proteins by Zic proteins. In addition, the physical interaction may contribute to the recruitment of both proteins onto the GLI-BS or core promoter, resulting in elevation of the local concentrations of both proteins. On the other hand, if we assume that the protein-to-protein association interferes with the ability of Gli or Zic to regulate transcription, then the presence of Zic would reduce the effect of Gli and vice versa. This could be the molecular basis of the Zic-Gli counteractivity. The presumptive molecular machinery of synergistic activation or inactivation by the Zic-Gli interaction may vary according to cell type and the developmental context.
Gli/Ci proteins function downstream of the hedgehog (hh) signaling pathway as both transcriptional activators and repressors (30 -33). On the other hand, the signal transduction pathway mediated by Zic proteins has not been well understood. Our results suggest that Zic proteins can interact with every Gli protein including the repressive form. We therefore speculate that Zic proteins are a potential modulator of the hh signaling pathway in various situations in animal development. Interestingly, expression of the Zic family grossly overlaps that of GLI3 in neural tube, somites, and limb buds (17,34,35). Further clarification of the role of Zic proteins in the hh signaling pathway should help clarify the molecular mechanisms of body pattern control.