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Human Molecular Genetics Advance Access originally published online on December 21, 2006
Human Molecular Genetics 2007 16(2):187-198; doi:10.1093/hmg/ddl461
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Nuclear import and export signals are essential for proper cellular trafficking and function of ZIC3

James E.J. Bedard1, Jennifer D. Purnell1 and Stephanie M. Ware1,*

1 Department of Pediatrics, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA

* To whom correspondence should be addressed at:, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 7020, Cincinnati, OH 45229, USA. Tel: +1 5136369427; Fax: +1 5136365958; Email: stephanie.ware{at}cchmc.org

Received November 16, 2006; Accepted December 6, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Missense, frameshift and nonsense mutations in the zinc finger transcription factor ZIC3 cause heterotaxy as well as isolated congenital heart disease. Previously, we developed transactivation and subcellular localization assays to test the function of ZIC3 point mutations. Aberrant cytoplasmic localization suggested that the pathogenesis of ZIC3 mutations results, at least in part, from failure of appropriate cellular trafficking. To further investigate this hypothesis, the nucleocytoplasmic shuttling properties of ZIC3 have been examined. Subcellular localization assays designed to span the entire open-reading frame of wild-type and mutant ZIC3 proteins identified the presence of nucleocytoplasmic transport signals. ZIC3 domain mapping indicates that a relatively large region containing the zinc finger binding sites and a known GLI interacting domain is required for transport to the nucleus. Site-directed mutagenesis of critical residues within two putative nuclear localization signals (NLSs) leads to loss of nuclear localization. No further decrease was observed when both NLS sites were mutated, suggesting that mutation of either NLS site is sufficient for loss of importin-mediated nuclear localization. Additionally, we identify a cryptic CRM-1-dependent nuclear export signal (NES) within ZIC3, and identify a mutation within this region in a patient with heterotaxy. These results provide the first evidence that control of cellular trafficking of ZIC3 is critical for function and suggest a possible mechanism for transcriptional control during left–right patterning. Identification of mutations in mapped NLS or NES domains in heterotaxy patients demonstrates the functional importance of these domains in cardiac morphogenesis and allows for integration of structural analysis with developmental function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mutations in members of the ZIC family of zinc finger transcription factors result in Dandy–Walker malformation, holoprosencephaly and X-linked heterotaxy (1–5), indicating a critical role for these genes in embryonic pattern formation. The family is characterized by C2H2 zinc finger DNA-binding domains, spanning over a third of the protein. The ZIC transcription factors (ZIC 1–5) share similarity with Drosophila odd-paired (opa) and members of the GLI protein family (6), with the greatest homology among superfamily members occurring within their zinc finger DNA binding domains. ZIC and GLI proteins bind identical cis elements, although GLI proteins bind with higher affinity. In addition, ZIC and GLI proteins physically interact through their zinc finger domains, suggesting that ZICs may function as co-activators with GLI (7,8).

The mammalian ZIC3 gene was first identified in the cerebellum of mice and has subsequently been shown to play a crucial role in early neural, neural crest and mesoderm development, as well as left–right axis formation (9–13). In humans, mutations or deletions of ZIC3 result in isolated congenital heart defects or heterotaxy, a phenotype with complex cardiovascular malformations and visceral situs anomalies (1, 3, 13–15). Previously, we developed transactivation and subcellular localization assays to test the function of ZIC3 point mutations (1). Mutations occurring between amino acids 268–323 of ZIC3 resulted in alteration of subcellular localization, leading to the hypothesis that mutations within this region disrupt cellular trafficking.

Nucleocytoplasmic shuttling is a mechanism which can regulate transcriptional activity by facilitating the cellular trafficking of transcription factors between the cytoplasm and nucleus. Nuclear import and export of transcription factors occurs by passive diffusion, active transport or by binding in a complex with an actively transported protein. Directed nuclear entry of a protein is determined by the presence of nuclear localization signals (NLSs) that recognize and associate with the nuclear import receptors. The NLS sites typically contain clusters of positively charged basic amino acids of lysine (K) and/or arginine (R). These form small consensus regions referred to as either ‘pat4’, ‘pat7’ or bipartite domains (16,17). ‘Pat4’ is composed of four K/R residues or three K/R and a histidine (H) or proline (P). ‘Pat7’ begins with a P and is followed within three amino acids by a basic segment containing three out of four K/R residues. The bipartite NLS contains two basic residues, followed by a 10 amino acid stretch, and three out of five additional basic residues. The NLS sites are recognized by the nuclear pore complex at the importin dock and enter into the nucleus (18). Directed nuclear exit of transcription factors is facilitated by the presence of nuclear export signals (NES), some of which bind to exportin (CRM-1). Consensus NES motifs are best characterized as hydrophobic and leucine-rich regions, but the precise site of interaction between the NES and CRM-1 receptor has not been determined (19,20).

To further understand how mutations in ZIC3 may result in aberrant subcellular localization, we have investigated the domains of ZIC3 required for nuclear import and export. Two NLS sites are identified in ZIC3, and inactivation of these sites reduces nuclear import. Furthermore, an encrypted CRM-1-dependent NES is identified. Patient mutations within the NLS and NES regions indicate the functional importance of these domains and suggest complex regulatory interactions within the zinc finger domains. These results represent the first evidence of nucleocytoplasmic trafficking in a member of the ZIC family of transcription factors and provide novel mechanistic information about the underlying etiology of heterotaxy and isolated congenital heart disease resulting from genetic alterations of ZIC3.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mapping of ZIC3 transport domains
In order to determine the regulatory domains involved in nucleocytoplasmic transport of the ZIC3 protein, experiments were performed using established shuttling assays to examine nuclear localization and nuclear export (21–23). The vector, pHM830, has been described previously and is constitutively cytosolic in the absence of an NLS (21). Generation of a C-terminal LacZ fusion protein increases the molecular weight of the construct, ensuring that fragments cannot passively diffuse into the nucleus. Overlapping fragments spanning the entire open-reading frame of ZIC3 were cloned in frame to generate eGFP–ZIC3–LacZ fusion constructs (Fig. 1A). Constructs were transfected into human epithelial-derived HeLa cells and murine NIH 3T3 cells in order to determine the critical regions of ZIC3 necessary for nuclear targeting. Representative immunofluorescence results are shown in Fig. 1B–G. Each construct was tested in both NIH 3T3 and HeLa cells and quantitative results for the former cell line are shown in Fig. 1H. Greater than 90% of full length ZIC3 (GFP 1–466) is nuclear, in agreement with our previously reported data using an epitope tagged cDNA (1). In contrast, all cells with the GFP 62–300 construct had cytosolic expression, with 80% being confined exclusively to the cytosol. Two constructs, GFP 290–466 and 290–420, were found within the nucleus in 82 and 95% of cells, respectively, suggesting that these protein domains contain elements required for nuclear localization. However, shorter GFP constructs of ZIC3 within amino acids 290–466 were predominantly expressed within the cytosol, indicating that a single strong NLS may not be present. Results obtained in HeLa cells were qualitatively identical with the exception of the construct GFP 290–350, which showed mixed nuclear and cytoplasmic localization in 39% of cells when compared with 20% identified in NIH 3T3 cells. Intracellular localization of ZIC3 fragments was further confirmed by western analyses performed using subcellular fractions (Fig. 1I). Representative examples using constructs GFP 250–300 and GFP 290–466 indicate predominantly cytosolic localization for the former and nuclear localization for the latter, in agreement with the immunofluorescence data. Probing with antibodies against GAPDH and c-Jun demonstrates the purity of the cytosolic and nuclear fractions.


Figure 4611
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  		Figure 1. Mapping the nuclear import domain of ZIC3. (A) Schematic of the pHM830 GFP-LacZ vector used for generating constructs. The amino acids of ZIC3 that are cloned in frame into pHM830 are indicated. (B–G) Representative GFP expression results in NIH 3T3 cells showing nuclear (B) and cytosolic (E) GFP expression. DAPI nuclear staining (C and F) and merged GFP and DAPI images (D and G) are shown for constructs GFP 290–466 and GFP 250–300. (H) Quantitative analysis of subcellular localization. Nuclear, mixed (nuclear and cytoplasmic) and cytoplasmic staining are indicated as percentages and are representative of at least 100 cell counts each from a minimum of three separate experiments. (I) Representative western blot analyses. Cells were transfected with GFP 250–300 and GFP 290–466 constructs and protein harvested as whole cell lysate or fractionated to obtain cytosolic or nuclear fractions and probed with the indicated antibody. C, cytosolic; N, nuclear; W, whole cell lysate.

 
Identification of multiple functional NLSs
ZIC3 was scanned for potential NLS domains. We found three putative NLS regions containing multiple positively charged amino acid residues (arginine, lysine and histidine) located at amino acids 263–270, amino acids 367–382 (hereafter NLS-1) and amino acids 403–410 (hereafter NLS-2) (Fig. 2A). Because the basic region at amino acids 263–270 lies outside the region mapped by deletion experiments (Fig. 1), we focused our experiments on the latter two basic regions. NLS-2 is highly homologous to a functional NLS in the zinc finger transcription factor PacC of Aspergillus nidulans (24). In order to test the function of these regions, the putative NLS sites were mutated within the context of an HA-ZIC3 cDNA expression construct. Site-directed mutagenesis was used to substitute nucleotides coding for lysine or arginine, as indicated (Fig. 2A). Subsequently, NLS-mutant plasmids were transfected into HeLa cells and subcellular localization was assessed by immunohistochemistry. Representative immunohistochemistry results are shown in Fig. 2B–M. In agreement with our GFP data, 90% of cells had exclusively nuclear localization of the wild-type ZIC3 protein (Fig. 2B–D and Q), whereas only 24% of ZIC3 NLS-1 mutants (Fig. 2E–G and Q) and 29% of NLS-2 mutants (Fig. 2H–J and Q) were exclusively nuclear.


Figure 4612
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  		Figure 2. Mapping of putative NLS sites in ZIC3. (A) Candidate NLS sites within the amino acids 290–466 region and identification of residues mutated via site-directed mutagenesis. The published PacC NLS site is shown in comparison with ZIC3 NLS-2. (B–M) Representative immunohistochemistry results. Subcellular localization of wild-type or mutant ZIC3 probed with anti-HA antibody (B, E, H, K), corresponding DAPI staining (C, F, I, L) and merged images (D, G, J, M). (N–P) Representative immunofluorescence results. Subcellular localization of GFP 367–382/403–410 construct showing nuclear GFP expression (N), corresponding DAPI staining (O) and merged image (P). (Q) Quantitative analysis of subcellular localization indicating percent nuclear, mixed and cytoplasmic. The pHM830 vector is included as a cytoplasmic control. Percentages are representative of at least 100 cell counts each from a minimum of three separate experiments.

 
Multiple NLS sites within a protein may act cooperatively in order to increase nuclear accumulation more efficiently (25). In order to evaluate a combinatorial function of the identified nuclear targeting domains, a second series of site-directed mutagenesis reactions were performed to generate mutations in both putative NLS sites within the context of HA-ZIC3 (double mutants). In the event of cooperative targeting by the NLS sites, decreased nuclear accumulation would be identified in the double mutants. However, the NLS-1,2 double mutant accumulated in the nucleus at levels similar to each independent mutation (Fig. 2K–M and Q), indicating that no additive effect occurs. Since NLS-1 and NLS-2 are closely arranged within zinc finger regions four and five of ZIC3, mutation of either NLS site might be sufficient for loss of importin-mediated nuclear localization.

In order to test the sufficiency of NLS-1 and NLS-2 for directing nuclear import, we created a heterologous protein containing the putative ZIC3 NLSs. The sequences coding for NLS-1 (amino acids 367–382) and -2 (amino acids 403–410) were inserted in frame into pHM830. Transfection of this construct, GFP 367–382/403–410, results in exclusively nuclear localization in 85% of cells, when compared with 0% in the pHM830 control (Fig. 2N–P and Q). Taken together with the site-directed mutagenesis results, these data indicate that NLS-1 and -2 are necessary and sufficient for nuclear import.

Nucleocytoplasmic shuttling in ZIC3 mutants
Treatment with leptomycin B (LMB), a specific inhibitor of the nuclear export receptor CRM-1, prevents the interaction of CRM-1 with the NES and results in the accumulation of NES containing proteins in the nucleus (20). We tested the ability of ZIC3 expression constructs to respond to LMB (Fig. 3) to determine whether patient mutations create or expose an NES. Both HeLa and NIH 3T3 cells were transfected with wild-type or mutant HA-ZIC3 and exposed to LMB for 12 h. Upon treatment of the cytoplasmic ZIC3 mutant T323M with LMB, specific retention in the nucleus was noted, suggesting the presence of an NES. In contrast, other cytoplasmic mutants such as H286R were unaffected by LMB treatment.


Figure 4613
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  		Figure 3. Nucleocytoplasmic shuttling in ZIC3 mutants. (A) Immunohistochemistry results with H286R and T323M ZIC3 mutants in the presence or absence of LMB. (B) Graphical representation of nuclear localization of mutant ZIC3 after 12 h exposure to LMB.

 
A time course analysis was performed to determine the sensitivity to inhibition of CRM-1-mediated export. The T323M mutant showed increased nuclear localization 4 h after the addition of LMB (data not shown). Progressive increases in nuclear accumulation continued until 12 h post-treatment, at which time ~80% of T323M was nuclear (Fig. 3B).

ZIC3 is an LMB-sensitive nucleocytoplasmic shuttling protein
The NES present in the T323M mutant could be newly created via the mutation since hydrophobic residues, including methionine, comprise an NES. Alternatively, the mutation may expose a cryptic NES present within the wild-type protein. To test the latter, we performed localization assays using ZIC3 constructs to determine whether an NES is present. Figure 4A shows that a fragment between amino acids 250 and 350 is sensitive to LMB treatment, indicating that an NES is present in the wild-type protein. We subsequently mapped this region to amino acids 290–350, consistent with the interval in which the mutation occurs (Fig. 4A and B).


Figure 4614
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  		Figure 4. ZIC3 is an LMB-sensitive nucleocytoplasmic shuttling protein. (A) Immunofluorescence results of pHM830 GFP-LacZ vector or indicated GFP-ZIC3 construct with or without 12 h of treatment with LMB. Nuclear accumulation in response to LMB is seen in GFP 250–350 and GFP 290–350, but not in pHM830 or GFP 250–300. (B) Quantitation of subcellular localization in ZIC3-GFP constructs (+/–12 h LMB treatment). (C) Nuclear localization of pHM840 vector, HA-ZIC3 (WT), GFP 290–466 and GFP 290–420 constructs with or without CRM-1 co-transfection. Error bars represent one standard deviation and asterisks indicate P-value of less than 0.05. (D) Amino acids 290–350 of ZIC3 followed by the consensus of leucine spacing and two well-described CRM-1-dependent NESs, HIV-1 REV and STAT1. Alignment of ZIC3 amino acid sequence to the consensus leucine-rich NES is shown with conserved amino acid residues in bold and mutations created by site-directed mutagenesis underlined. (E) HA-ZIC3 (WT) and full-length NES-1–3 mutants were transfected with or without CRM-1. The percent difference in cytoplasmic accumulation in CRM-1 co-transfected cells is shown for each construct. Experiments were performed in triplicate with a minimum of 100 cells counted per experiment per construct. (F) Quantitative analysis of the subcellular localization of GFP 290–350 and GFP 290–350 NES mutant fragments. Nuclear accumulation occurs in NES mutants. Results obtained after 12 h treatment with LMB or co-transfection with CRM-1 are indicated. (G) Transcriptional activation of HA-ZIC3 (WT), NLS-1, NLS-2 and NES-3 mutants. Cells were co-transfected with the indicated constructs, TK Renilla luciferase and a Gli binding site luciferase reporter. Fold activation was calculated relative to the reporter and is expressed as a percentage compared with that of WT ZIC3. Error bars represent one standard deviation and are the results of three independent experiments.

 
Overexpression of CRM-1 has previously been shown to induce nuclear export of NES-containing proteins that are localized to the nucleus (26). Therefore, to further confirm the results obtained with LMB treatment, we tested the ability of CRM-1 to alter subcellular localization of ZIC3. In the presence of a functional CRM-1-dependent NES, co-transfection should result in decreased nuclear localization due to increased transport through the nuclear pore. pHM840, HA-ZIC3, GFP 290–466 or GFP 290–420 were transfected independently or in combination with CRM-1. The pHM840 control vector is designed to localize to the nucleus and does not have an NES. Following transfection, cells were processed for immunohistochemistry or GFP detection (Fig. 4C; data not shown). Overexpression of CRM-1 results in statistically significant relocation of ZIC3 from the nucleus to the cytoplasm in all three ZIC3 containing constructs examined, but not in the pHM840 control. The magnitude of export was similar between ZIC3 containing constructs. These results provide independent evidence that CRM-1 is the export receptor for ZIC3.

The nuclear export receptor CRM-1 recognizes hydrophobic leucine-rich motifs. Comparison of the amino acid sequence of ZIC3 within amino acids 290–350 with the consensus CRM-1-dependent NES is shown in Figure 4D. A single region with hydrophobic amino acids was identified in ZIC3 (amino acids 313–325), although this region is not highly homologous with the consensus NES. Site-directed mutagenesis was used to alter one, two or four critical hydrophobic amino acids within the region in the context of both the full length ZIC3 protein and the GFP 290–350 construct (Fig. 4D). Loss of an NES should result in increased retention in the nucleus, and/or loss of responsiveness to LMB or CRM-1. First, the full length ZIC3 constructs were tested for responsiveness to co-transfection with CRM-1. We observed a 35% increase in cytoplasmic accumulation in the wild-type protein when co-transfected with CRM-1 (Fig. 4E; see also results in 4C), but only modest cytoplasmic accumulation in the NES-1 mutant and no accumulation in either the NES-2 or -3 full-length ZIC3 mutants (Fig. 4E). These results suggest that mutation of residues within amino acids 290–350 block exportin-mediated nuclear export. To further evaluate the putative NES, we examined nuclear accumulation and LMB responsiveness in the context of the amino acids 290–350 fragment. Subcellular localization assays demonstrate significant increases in nuclear accumulation when one, two or four amino acids of the putative NES were altered, suggesting that site-directed mutagenesis inhibits nuclear export. In comparison with the wild-type ZIC3 fragment, a significant loss of responsivenss to LMB occurred when one or two hydrophobic residues were mutated (NES-1 and NES-2), and responsiveness to LMB was completely abolished when all four were mutated (NES-3). In contrast to wild-type ZIC3, which is exported upon co-transfection with CRM-1, NES-3 showed no decrease in nuclear localization with CRM-1 overexpression (Fig. 4F). These data indicate that the missense mutations introduced into the hydrophobic region of GFP 290–350 significantly inhibit CRM-1-dependent nuclear export in HeLa cells.

Taken together with previously published data on DNA binding, these results indicate that the zinc finger regions of ZIC3 contain motifs necessary for DNA binding, nuclear import and nuclear export. In order to determine whether the NLS and NES mutants retain transcriptional activating function, we used a multimerized Gli binding site reporter and luciferase assays. A decrease in transactivation was noted in both NLS-1 and NLS-2 mutants when compared with wild-type (Fig. 4G). However, our subcellular localization results indicate that these constructs are primarily cytoplasmic (Fig. 2Q), therefore some decrease in transcriptional activation is anticipated. NES-3 retains significant transcriptional activation function. These results extend the structure-function analyses by suggesting that NLS and NES mutants retain some degree of transcriptional activation function.

Conservation of structural domains in ZIC3
Given the importance of ZIC proteins in organogenesis, we wished to determine whether the structural domains identified in this study are conserved between orthologs and paralogs. Figure 5 indicates that the regions containing the putative NLSs and NES are completely conserved between the species examined. Table 1 shows that the motifs are also highly conserved between family members. The identified domains were completely conserved in ZIC1 and ZIC2 and greater than 87% conserved in ZIC4 and ZIC5. While the importance of these highly conserved domains for nuclear trafficking requires testing in other ZIC family members, these results suggest that control of the subcellular localization of ZICs may serve as a general mechanism for functional control.


Figure 4615
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  		Figure 5. Interspecies comparison of ZIC3 protein sequences. Zinc finger domains (ZF-1–5) are indicated in bold and NES (amino acids 290–350) and NLS regions are highlighted by light and dark grey boxes, respectively. (*) indicates identical, (:) functionally conserved or (.) semi-conserved amino acid residues. Sequences were aligned using the ClustalW (1.83) multiple sequence alignment program from European Bioinformatics Institute.

 


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  		Table 1. Homology of ZIC proteins within identified import and export domains of ZIC3

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
ZIC proteins are important mediators of developmental patterning and mutations in ZIC1–4 cause congenital anomalies including Dandy–Walker malformation (ZIC1 and ZIC4), holoprosencephaly (ZIC2), heterotaxy (ZIC3) and congenital heart defects (ZIC3). However, very little is known about the biochemical properties or cellular trafficking of these zinc finger proteins.

In vitro investigations indicate that ZIC family proteins are nuclear (8). However, we have previously shown that specific mutations in patients with heterotaxy alter the subcellular localization of a ZIC3 expression construct (1). In addition, ZIC1, -2 and -3 exhibit cytoplasmic localization upon co-expression with the inhibitor of MyoD family protein (27). Taken together, these studies suggest that nuclear import of ZIC3 is actively regulated. One limitation of in vitro subcellular localization assays is that overexpression may abrogate normal protein interactions. Therefore, we have examined the endogenous expression of ZIC3 in the HeLa and NIH 3T3 cell lines used in this study (Supplementary Material, Fig. S1). The results demonstrate that ZIC3 is not constitutively nuclear in these cell lines and are consistent with a requirement for nuclear import and export.

In this study, we investigate the cellular trafficking properties of ZIC3 and demonstrate that it is a nucleocytoplasmic shuttling protein that can actively enter and exit the nucleus. We demonstrate that a region containing the zinc finger DNA-binding domain is necessary and sufficient for transport into the nucleus in a cellular trafficking assay. We further define two NLS signals within this fragment, which we have mapped to amino acids 367–382 and 403–410 (Fig. 6), and demonstrate that mutation of these signals significantly disrupts nuclear import. NLS-2 is highly homologous to a functional NLS in PacC. Both NLSs have two basic residues followed by spaced zinc coordinating histidines. Functional analysis of this NLS using genetic selection in Aspergillus indicates that the first basic residue is critical for both nuclear import and DNA binding, whereas the second basic residue functions only in nuclear import (24).


Figure 4616
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  		Figure 6. (A) Schematic representation of the ZIC3 gene and (B) partial tertiary structure model of ZIC3 protein (amino acids 264–382) based upon current predictive homology comparison with similar proteins. Locations of NLS and NES sites, zinc fingers and human patient and site-directed mutations indicated. DeepView/Swiss-Pdb Viewer (v3.7) software was used to generate the 3D model.

 
Two lines of evidence indicate that ZIC3 undergoes nuclear export by a CRM-1-dependent mechanism. First, the data demonstrate that cytosolic fragments of ZIC3 are retained in the nucleus after treatment with the export inhibitor LMB. Secondly, co-expression of both full length wild-type ZIC3, as well as GFP-ZIC3 deletion constructs, with CRM-1 results in the relocation of ZIC3 to the cytoplasm. Identification of NLS and NES domains provides a mechanistic understanding of the cellular trafficking of ZIC3 and may be important for further delineation of its developmental pathogenesis.

Analysis of known ZIC3 patient mutations in the context of the identified structural domains reveals several important features (Fig. 6). First, many of the ZIC3 patient mutations seem to congregate at, or in close proximity to, the first zinc finger region (aminio acids 253–287). The amino acid sequence of this zinc finger may represent a more recent evolutionary event since it is the least conserved zinc finger of ZIC3 (Fig. 5) (1,28). In addition, on the basis of the crystal structure of GLI, it is possible that the first zinc finger of ZIC3 may not contact DNA (29). Missense mutations within this region might therefore cause a less severe phenotype, resulting in increased survival rate and increased ascertainment of mutations. Data from Zic3 deficient mice indicate a high percent of gastrulation defects prior to left–right patterning, suggesting that complete loss of ZIC3 function may result in early miscarriage in many cases (30). Mutations surrounding the first zinc finger that alter tertiary protein structure would likely leave the DNA-binding domain and NLS-1 and -2 unaffected, thus allowing for partial function. In vitro, mutation of the first zinc finger of ZIC3 results in only partial inhibition of nuclear translocation [(1); unpublished data].

Secondly, examination of the tertiary structure of ZIC3 illustrates that mutations known to cause loss of nuclear localization cluster near NLS and NES domains (Fig. 6B). C268X and Q292X are both truncating mutations that result in loss of mapped NLS domains. These construct both have cytoplasmic localization in vitro, further substantiating the importance of mapped NLSs in nuclear import. However, simply making amino acid substitutions within the zinc finger domains does not result in loss of nuclear localization, since several ZIC3 point mutations within the zinc finger domains retain nuclear localization (1). This strongly suggests that the positively charged residues within the NLS sites function as a major control mechanism for cellular trafficking of ZIC3. Thus, results of subcellular localization studies performed with mutant ZIC3 proteins are in agreement with the mapped NLS domains identified in this study.

NLS, NES and DNA-binding domains of ZIC3 overlap. This is a relatively common finding among zinc finger proteins, also being seen in Smad4 and PacC (24,31). Previous studies have indicated the difficulty of dissection of import and export motifs from DNA-binding domains due to functional overlap (24,32–35). It has been proposed that NLSs and DNA-binding domains co-evolve, since active nuclear entry and DNA binding are highly interdependent (32) and overlap of these motifs may aid release of a protein from cognate importers. The delineation of specific import and export motifs within the zinc finger binding domains of ZIC3 provides a basis for further investigation of the requirement for nuclear localization during development.

Despite the absence of a traditional NES site, we were able to map a CRM-1-dependent NES domain centered around amino acids 313–325. Our data indicate that this NES site is hidden and structural changes in ZIC3, such as the introduction of missense mutations, are required to expose the motif and facilitate export. As shown in Figs. 5 and 6, both NLSs and NES overlap with the zinc fingers, which have previously been shown to bind GLI. It has been shown previously that co-expression of ZIC1 and GLI1 resulted in increased nuclear translocation and transcriptional regulation through a protein–protein association (7). GLI transcription factors contain a functionally active bipartite NLS motif as well as an NES, and undergo complex nucleocytoplasmic trafficking in conjunction with Suppressor of fused to mediate hedgehog signaling (36–41). Further investigation is required to determine whether physical interactions between ZIC3 and GLI results in the coordination of several NLS sites for recruitment to the nucleus and/or alteration in exposure of NES motifs. Studies investigating possible binding partners, including GLI family members, may provide additional clues for further understanding the nucleocytoplasmic shuttling mechanism of ZIC3 during left–right patterning and cardiac development.

While nucleocytoplasmic trafficking is a well-described regulatory mechanism for control of cell-cycle progression and proteins involved in DNA damage (42–44), it remains a relatively unexplored mechanism for regulation during development (45). Transcriptional activation and repression must be precisely controlled in a temporal and tissue-specific manner for proper body pattern formation. Control of nuclear import and export is thus an attractive mechanism to balance transcriptional activity during organogenesis, and future work will be necessary to determine the tissue-, developmental-, and cell-cycle-specific regulation of ZIC3. Members of the SOX family of transcription factors have recently illustrated the importance of proper subcellular localization for normal development (46–49), with defects in cellular transport of SOX members underlying sex reversal (SRY, SOX9), campomelic dysplasia (SOX9) and defects in transcriptional activation (SOX10). In analogy to the SOX family, control of nuclear import and export may be a general mechanism of transcriptional regulation of GLI superfamily members, including ZIC3.

In summary, we have identified novel and functional NLSs and NES in ZIC3 and demonstrate the importance of these sites for cellular trafficking. Identification of mutations in mapped NLS or NES domains in patients with heterotaxy or congenital heart disease demonstrates the importance of these domains in cardiac morphogenesis and allows for integration of structural analysis with developmental function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cell culture and conditions
HeLa and NIH 3T3 cells were maintained in Dulbecco's Modification of Eagle's Medium (Cellgro no. 10-017-CV) supplemented with 10% fetal bovine serum (FBS) and Minimal Essential Medium (Gibco no. 41500-034) supplemented with 1 mM sodium pyruvate and 10% FBS, respectively, at 37°C and 5% CO2. LMB was purchased from Sigma. For LMB treatments, 5 ng/ml LMB was added the day after transfection and incubated for times described in the text.

Expression constructs
Construction of the wild-type hemagglutinin (HA)-ZIC3 and HA-ZIC3-mutant expression vectors have been described previously (1). For NLS mutants, the oligonucleotides used for site-directed mutagenesis were: KK376AA-F (5'-TGC CAA CAG CAG CGA CCG TGC AGC GCA CAT GCA TGT GCA TAC CT-3'), KK376AA-R (5'-AGG TAT GCA CAT GCA TGT GCG CTG CAC GGT CGC TGC TGT TGG CA-3'), R404A-F (5-GCA CCC GAG CTC CCT GGC AGA ACA CAT GAA GGT TC-3'), R404A-R (5'-GAA CCT TCA TGT GTT CTG CCA GGG AGC TCG GGT GC-3'), where the mismatches are underlined. NLS-2 utilized HA-ZIC3 K405E, a previously described expression construct based on a patient mutation, as the template for site-directed mutagenesis. For NES mutants, the oligonucleotides used were: LV315AA-F (5'-CTT TCA AGG CGA AGT ACA AAG CCG CCA ACC ACG CAC GAG TGC AC-3'), LV315AA-R (5'-GTG CAC TCG TGC GTG GTT GGC GGC TTT GTA CTT CGC CTT GAA AG-3'), I319A-F (5'-GTA CAA ACT GGT CAA CCA CGC TCG AGT GCA CAC GGG CGA G-3'), I319A-R (5'-CTC GCC CGT GTG CAC TCG AGC GTG GTT GAC CAG TTT GTA C-3'), LV315AA-I319A-V321A-F (5'- CAA AGC GGC CAA CCA CGC ACG AGC GCA CAC GGG CGA GAA GC-3'), LV315AA-I319A-V321A-R (5'-GCT TCT CGC CCG TGT GCG CTC GTG CGT GGT TGG CCG CTT TG-3'). Mutagenesis reactions were carried out using the QuikChange Site-Directed Mutgenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations. In each PCR reaction, 50 ng of template DNA was used. The pHM830 (GFP-LacZ) and pHM840 (GFP-NLS-LacZ) plasmids were gifts from Dr Ray Truant. ZIC3 fragments generated by PCR using human ZIC3 cDNA as the template were inserted in frame into the SacII-XbaI sites of pHM830. The heterologous protein construct containing the two putative ZIC3 NLS motifs (NLS-1 and -2) was created by generating a double stranded DNA oligonucleotide containing the NLS sequences and inserting it in frame into the SacII-XbaI sites of pHM830. All expression constructs were verified by sequencing. Sequences of oligonucleotides used for PCR amplification of ZIC3 fragments are available upon request.

Immunohistochemistry and subcellular localization
Cells were plated at a density of 1 x 105 on 20 x 20 mm2 glass coverslips 1 day prior to transfection. For GFP analyses, cells were washed in cold phosphate buffered saline (PBS) twice, fixed in ice cold 4% paraformaldehyde/PBS on ice for 30 min, rinsed in PBS three times and mounted on slides with DAPI. Immunohistochemistry and subcellular localization assays were performed as previously described (1) using an anti-HA rabbit polyclonal antibody (Novus Biologicals) at 1 : 250 dilution and Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes) secondary antibody at a dilution of 1 : 500. For detection of endogenous ZIC3, a goat anti-ZIC3 polyclonal antibody (Santa Cruz # sc-28156) was used at 1 : 50 dilution and Alexa Fluor 488 rabbit anti-goat IgG (Molecular Probes) secondary antibody was used at a dilution of 1 : 500. For subcellular localization assays, all experiments were performed in triplicate with at least 100 cells counted.

Luciferase assays
The luciferase assays were carried out as previously described (1). Briefly, HeLa cells were transfected using Lipofectamine Plus (Invitrogen) according to the manufacturer's protocol. Co-transfections were performed using HA-ZIC3 constructs and 12Gli-RETKO-luciferase, a reporter with 12 multimerized Gli binding sites. Cells were harvested 48 h after transfection, and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. The relative fold activation was calculated in comparison with transfection of 12Gli-RETKO-luciferase with empty vector. Results represent three independent transfection experiments and error bars represent one standard deviation.

Western analyses
Cells were washed in ice-cold PBS and harvested in lysis buffer containing 50 mM Tris (pH 7.5), 250 mM sodium chloride, 3 mM EDTA, 3 mM EGTA, 1% Tween X-100, 0.5% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, 30 µg/ml aprotinin. Whole cell lysate was freeze-thawed and vortexed briefly, followed by centrifugation (12 000g) at 4°C for 10 min to pellet cellular debris. Nuclear and cytoplasmic fractionated protein samples were isolated using the Paris protein and RNA isolation system (Ambion) as per manufacturer's instructions. Protein levels were quantified by the method of Lowry et al. (50). (Bio-Rad). Twenty-five µg of total protein per sample was electrophoresed on 10% Tris–glycine gels (Invitrogen). Gels were transferred to pre-soaked 0.45 µm nitrocellulose membranes (Invitrogen) using the Bandit Tank Electroblotting System (Owl) following manufacturer's recommendations. Western blots were blocked in 5% non-fat dry milk in 1 x tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h followed by incubation with a 1 : 5000 dilution of anti-LacZ (Abcam # ab616). Blots were subsequently washed three times, 20 min each, in 1 x TBST, followed by 1 h incubation in 5% milk in TBST with a 1 : 10 000 dilution of goat anti-rabbit HRP linked secondary antibody (Santa Cruz no. sc-2054). After three additional 20 min washes in 1X TBST, blots were incubated for 5 min in ECL Plus Detection Reagents (Amersham) and developed on HyBlot CL autoradiography film (Denville Scientific).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors thank Dr Ray Truant for providing the pHM830 and pHM840 plasmids, Dr Gerard Grosveld for providing the CRM-1 plasmid and Dr Rune Toftgard for the gift of the 12Gli-RETKO-luciferase reporter. This work was supported by grants from the NIH (HL6735) and American Heart Association (0555279B) to S.M.W.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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