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...

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled‐coil domain, a novel conserved domain (DSPRC) and a C‐terminal hydrophobic region that is predicted to be a transmembrane domain. N‐terminally epitope tagged‐Rines (Nt‐Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt‐Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt‐Rines is an integral membrane protein with most of its N‐terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt‐Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt‐Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin–proteasome pathway was further supported by its binding to the UbcH6 ubiquitin‐conjugating enzyme and by its trans‐ubiquitination enhancing activities. These results suggest that Rines is a membrane‐bound E3 ubiquitin ligase.