Physical and Functional Interactions between Zic and Gli Proteins

Abstract

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) (1-3). 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. EXPERIMENTAL PROCEDURESPlasmids-To express Flag-tagged and hemagglutinin (HA) 1 -tagged proteins, the relevant sequence was amplified by PCR, verified by DNA sequencing, and subcloned into pCMVtag2 (Stratagene) and pcDNA3HA (a gift from Dr. T. Nakajima). Sequences cloned included the full-length mouse Zic1-(1-447) and mouse Zic2 and Zic3 (19,21). The deletion seri...

Cerebellar development and disease

“…These mutants provide the first model to delineate the pathogenesis of DWM. Since Zic proteins interact with Gli proteins, which are obligate downstream components in Shh signal reception, it has been hypothesized that Zic proteins modulate Shh signaling [55] [56,57] and may be involved in Shh-regulated EGL proliferation. The role of Zic4 is not well understood, but Zic1 activity is required to maintain EGL cells in a progenitor state [57,58], although a direct role in the reception of Shh signaling has not been established.…”

Emerging roles for zic genes in early development

“…However, C 2 H 2 zinc finger domains are not generally bifunctional with respect to DNA binding and protein-protein interactions (Pabo et al, 2001;McCarty et al, 2003). Nevertheless, in cell-based assays, the zinc finger domains of Zic proteins are reported to form heterodimers with Gli proteins (Koyabu et al, 2001), which are also C 2 H 2 zinc finger transcription factors. Specifically, zinc fingers 3-5 of Zic recognize zinc fingers 3-5 of Gli (Koyabu et al, 2001).…”

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…As discussed in greater detail in section 1.3.2, fingers 2-5 bind DNA (Pavletich and Pabo, 1993). Fingers 3-5 mediate a PPI with multiple members of the Zic family of proteins; however, neither the contribution of individual fingers nor the binding surface has been elucidated (Koyabu et al, 2001). …”

“…As exemplified by the Ikaros family of proteins, in which 4 of the 6 C2H2 domains bind DNA and the remaining 2 bind protein, individual or clusters of C2H2 domains within a single protein may interact with multiple types of biomolecules (Hahm et al, 1998;Molnar and Georgopoulos, 1994;Morgan et al, 1997;Perdomo et al, 2000). In addition, individual fingers can separately interact with multiple types of biomolecules, as in the case of Gli proteins which use F3-5 to interact with both DNA and protein, but probably not at the same time (Koyabu et al, 2001). Finally, as seen in Zac1 finger 2, individual fingers can also interact with both protein and DNA at the same time (Hoffmann et al, 2006 (Bairoch and Apweiler, 1997;Sonnhammer et al, 1998).…”