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Previous Article | Next Article 
Molecular and Cellular Biology, August 2002, p. 5826-5834, Vol. 22, No. 16
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.16.5826-5834.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Institut für Biochemie,1 Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany2
Received 17 January 2002/ Returned for modification 26 February 2002/ Accepted 8 May 2002
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ABSTRACT |
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INTRODUCTION |
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One of the best characterized groups of Sox proteins is subgroup E. This group includes Sox8, Sox9, and Sox10. Sox8 is the most recently found member of this group, and its exact function is still unknown (34, 42, 43). Sox9 is essential for chrondrocyte differentiation and cartilage formation in mouse (2) and chick (19). In accord, inactivation of a single Sox9 allele in humans is the cause of a severe skeletal malformation syndrome named campomelic dysplasia (15, 49). In XY individuals, campomelic dysplasia is often linked to sex reversal. Sox10, finally, is essential for neural crest development and has been identified as the causative gene for Hirschsprung-Waardenburg syndrome (20, 23, 35, 44). Inactivation or deletion of both Sox10 alleles in mice leads to a complete loss of neural crest-derived melanocytes, the enteric nervous system, and all glia in the peripheral nervous system (6). Additionally, terminal differentiation of oligodendrocytes is disturbed (46). Target genes of Sox10 include genes important for glial development (ErbB3) and identity such as the genes for the myelin proteins protein zero (P0), myelin basic protein, and proteolipid protein (PLP) (6, 31, 46).
One defining feature of eukaryotic cells is their spatial and functional division into nucleus and cytoplasm by the nuclear envelope. This separation requires specific mechanisms for the continuous transport of a large number of macromolecules between both compartments. Nucleocytoplasmic transport takes place through the nuclear pore and is highly regulated by specific signals and transport receptors. In general, nuclear import (reviewed in references 18 and 30) requires energy and the presence of importin 
, importin ß, RanGTP, and short stretches of basic amino acids, termed nuclear localization signals (NLS), in the cargo (4, 9). Sox9 contains two NLS within the HMG domain (47). These signals are highly conserved in Sox8 and Sox10. In contrast, the signal-mediated nuclear export pathways are less understood. The best-characterized nuclear export signal (NES) is the small, hydrophobic, leucine-rich NES, which was initially described in the protein kinase inhibitor (51) and in the human immunodeficiency virus type-1 (HIV-1) Rev protein (13). The direct interaction with the export factor CRM1 is absolutely essential for the translocation of proteins containing a leucine-rich NES from the nucleus into the cytoplasm (14, 16, 45). Leucine-rich NESs have been identified in an increasing number of cellular and viral proteins executing heterogenous biological functions. Those include RNA transport (22, 24, 37), cell cycle (48), regulation of kinase activity (12), and transcriptional control (1, 17). Proteins containing both NLS and NES have the capacity for continuous shuttling between the cytoplasm and the nucleus. In the case of transcription factors this is of particular interest, because of the possibility of further regulation of transcription factor activity. So far, nuclear export has not been analyzed in any Sox protein. Here we investigate the capacity of Sox10 to shuttle between the nucleus and the cytoplasm and analyze potential consequences for its function as transcriptional regulator.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NES and pEGFP-Sox10-NES-L138A express GFP fusion proteins in which the leucine residues corresponding to Sox10 amino acid positions 134 and 138 or 138 alone were mutated into alanines. In pEGFP-NESREX-Sox10-NES-L138A the NES (amino acids [aa] 82 to 93) (11) of human T-cell leukemia virus type 1 Rex was fused in frame between GFP and Sox10-NES-L138A. A bacterial vector expressing GST-Sox10-NES-GFP wild-type protein was generated by insertion of a double-stranded synthetic oligonucleotide between the BamHI and NheI site of pGEX-GFP (39). The oligonucleotide encodes a short peptide motif (Sox10 aa 134 to 145) fused in frame between glutathione S-transferase (GST) and GFP. pGEX-Sox10-NES-GFP and pGEX-Sox10-NES-L138A-GFP express mutated versions of this fusion protein in which the leucine residues corresponding to Sox10 aa 134 and 138 or 138 alone were mutated into alanines. Plasmids GST-Rev-NES-GFP (38), pCFN-ß-galactosidase (ß-Gal), pCFNrev-ß-Gal (41), the pUL69 expression plasmid (24) and the luciferase reporter plasmid carrying the rat P0 promoter from position -915 to +48 are described elsewhere (31). Cell culture, transfection, luciferase assays, RNA preparation, semiquantitative reverse transcription (RT)-PCR, Western blot analysis and EMSA. HeLa cells, C6 cells and N2A neuroblastoma cells were maintained and transfected with calcium phosphate as described (31, 38). For luciferase assays, N2A neuroblastoma cells were transfected in triplicates on 35 mm dishes with 0.2 µg of luciferase reporter plasmid and 1 µg of the respective Sox10 construct. After transfection cells were returned to Dulbecco's modified Eagle medium containing 4% fetal calf serum. Cells were harvested 36 h posttransfection, and extracts were assayed for luciferase activity (32). Stable N2A cells capable of expressing Sox10 in a tetracycline inducible manner were kept, treated with doxycycline, and used for RNA preparation as described (31). Detection of products specific for P0, PLP, and GAPDH in cDNA generated by reverse transcription of RNA from these cells was performed by semiquantitative PCR (31, 46). Where indicated, cells were treated with 2.5 ng/ml LMB (kindly provided by Minoru Yoshida, Tokyo, Japan) for 24 h. For Western blot analysis whole cell extracts were prepared and proteins were separated on 10% SDS-polyacrylamide gels. After transfer to nitrocellulose membranes (Optitran-B85; Schleicher and Schuell, Dassel; Germany), Western blots with antibodies against GFP (Clontech Laboratories, Palo Alto, Calif.) and Sox10 (23) were performed as described (40). The same extracts were incubated with the 32P-labeled Sox10 binding site C/C' as described (31).
Purification of GST-GFP fusion proteins, microinjection and homokaryon analysis. GST-GFP hybrid proteins were expressed in Escherichia coli BL21 and purified on glutathione Sepharose 4B (Pharmacia Biotech, Germany) as previously described (38). Eluted proteins were analyzed by SDS-PAGE and Coomassie blue staining. Fractions were pooled, concentrated by ultrafiltration with a Nanosep filter (Pall-Filtron, USA) and microinjected into the nuclei of HeLa or Vero cells at 1.5 mg/ml in combination with rabbit IgG (1.0 mg/ml) (injection control) by using a CompiC INJECT computer-assisted injection system (Cellbiology Trading, Hamburg, Germany). Cells were fixed 45 min postinjection with 3% paraformaldehyde, and the injected proteins were analyzed by immunofluorescence analysis as described previously (39). In some experiments, 2.5 ng/ml LMB was added 2 h prior microinjection. For homokaryon analysis, HeLa cells were incubated for 24 h in MEM containing 1% bovine serum albumin. Homokaryons containing two nuclei were microinjected with full-length GFP-Sox10 fusion proteins (prior isolated from GFP-Sox10 overexpressing cells) together with a rabbit IgG into one nucleus. After 2 h of incubation at 37°C, cells were processed for immunofluorescence analysis.
Heterokaryon assays. Nucleocytoplasmic shuttling of Sox10 and its mutants was analyzed in a heterokaryon assay, as initially described by Pinol-Roma and Dreyfuss (36). Briefly, transfected HeLa cells or C6 cells were seeded on glass coverslips together with an equal number of NIH 3T3 cells and were incubated overnight. Cells were incubated with 50 µg/ml cycloheximide 30 min prior to the fusion and throughout the experiment. In some experiments, 2.5 ng/ml LMB was added 3 h before cell fusion. Subsequently, cells were washed in PBS, and heterokaryons were formed by incubating the cells for 2 min in 50% polyethylene glycol 8000 in Dulbecco's modified Eagle medium. Cells were returned to fresh medium containing cycloheximide (50 µg/ml) and LMB (2.5 ng/ml) when needed. After 2 h incubation at 37°C, cells were processed for indirect immunofluorescence analysis. After secondary antibody incubation, Hoechst 33258 (5 µg/ml; Sigma, Taufkirchen, Germany) were added for 5 min. At least 25 heterokaryons were analyzed for each experiment and nuclear shuttling was only scored positive when a minimum of 80% of the heterokaryons showed a positive staining of the investigated protein.
Immunofluorescence microscopy. Immunofluorescence microscopy was performed essentially as previously described (40). Cells were fixed for 20 min with 3% paraformaldehyde. Subsequently, cells were incubated with 0.1 M glycine, pH 8.5 for 15 min and blocked for 30 min with 1% bovine serum albumin-phosphate-buffered saline (PBS). Cells were then stained for 30 min with the primary antibodies and after extensive washing with PBS further incubated with appropriate secondary antibodies coupled to Cy2 or Cy3 (Biotrends, Cologne, Germany) for 30 min. The samples were washed in PBS, mounted in Mowiol (Calbiochem, La Jolla, Calif.) and analyzed using a Leica inverted microscope. Images were recorded with a cooled MicroMax charge-coupled device camera (Princeton Instruments, Stanford, Calif.) and processed using the IPLab spectrum and Adobe Photoshop software packages.
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RESULTS |
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Sox10 contains an active leucine-rich NES. The most common NES are characterized by small, hydrophobic, leucine-rich regions (13). Sox10 contains a similar sequence between aa 134 and 145. To determine whether this sequence indeed acts as a NES, we inserted the aa 134 to 145 of Sox10 between glutathione S-transferase (GST) and GFP. For a positive control we used a fusion protein containing the NES of HIV-1 Rev. The recombinant fusion proteins were purified from E. coli and microinjected into the nuclei of Vero cells together with rabbit immunoglobulin G (IgG) as a marker for the injection site. At 45 min after injection, the cells were fixed and immunostained for the coinjected IgG control. The injected rabbit IgG was detected exclusively within the nucleus (Fig. 4A, C, E, and G), whereas the GST-GFP fusion proteins, containing either the NES of Sox10 (Fig. 4B) or Rev (Fig. 4F), were translocated from the nuclear injection site to the cytoplasm in significant amounts. This indicates that the Sox10 signal sequence is transferable and can function as an autonomous NES.
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To further characterize the Sox10 nuclear export pathway, the NES of Sox10 was mutated. pGEX-Sox10-NES-GFP and pGEX-Sox10-NES-L138A-GFP express mutated versions of the GST-Sox10-NES-GFP in which the leucine residues corresponding to Sox10 amino acid positions 134 and 138 (Sox10NES) or 138 alone (Sox10-L138A) were exchanged to alanines. The fusion proteins were microinjected into the nuclei of Vero cells together with the control rabbit IgG. One hour after injection, the cells were fixed and immunostained for the coinjected IgG control. In sharp contrast to the GST-GFP fusion protein containing the wild-type NES of Sox10, both fusion proteins containing mutated versions of the Sox10-NES were not able to leave the nucleus (Fig. 4J and L) and remained colocalized with the coinjected rabbit IgG (Fig. 4I and K).
To directly investigate the contribution of the general export factor CRM1 on the subcellular distribution of Sox10, C6 cells were transfected with a CRM1 expression plasmid and subsequently immunostained for the presence and localization of CRM1 and Sox10. Overexpression of CRM1 (Fig. 3M) resulted in a partial relocalization of endogenous Sox10 from the nucleus into the cytoplasm (Fig. 3N), providing independent evidence that CRM1 is the physiological export receptor for Sox10.
The leucine-rich NES of Sox10 mediates nucleocytoplasmic shuttling of Sox10.
To analyze the activity of the leucine-rich NES in the context of the full-length Sox10 protein, we introduced the leucine-to-alanine mutations at aa 134 and 138 into the GFP-Sox10 fusion protein. In control heterokaryon assays, GFP-Sox10 wild-type fusion protein was rapidly translocated from the HeLa donor nucleus into the surrounding acceptor nuclei (Fig. 5A), and LMB significantly reduced this translocation (Fig. 5C). Whereas LMB treatment interfered with shuttling of GFP-Sox10 (Fig. 5H), it did not prevent translocation of the CRM1-independent pUL69 (24) in the same heterokaryon (Fig. 5G), proving that cells under LMB treatment were viable and that fusion and mixing of cytoplasm had occurred. The introduction of the leucine-to-alanine mutations in GFP-Sox10 strongly reduced the ability to shuttle from the donor nucleus into the surrounding acceptor nuclei (GFP-Sox10-NES, Fig. 5E), although it did so less efficiently than LMB treatment. The respective Hoechst 33258 counterstains demonstrate that the cell density was comparable in all cases (Fig. 5B, D, F, and I). We conclude that the identified leucine-rich export signal is a major determinant for the nucleocytoplasmic shuttling ability of Sox10. The presence of an additional weak export signal cannot be ruled out at present.
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NES together with a rabbit control IgG into only one nucleus. After 2 h of incubation at 37°C, the injected rabbit IgG was detected exclusively within the injected nuclei (Fig. 6A and D), whereas the GFP-Sox10 wild-type fusion protein underwent a significant translocation from the injected nucleus into the second uninjected nucleus (Fig. 6B). In contrast, the mutated GFP-Sox10NES fusion protein was not able to cross the nuclear pore of the injected nucleus and was not found in the potential acceptor nucleus within the same cell (Fig. 6E).
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NES and GFP-Sox10L138A were reduced in their ability to stimulate gene expression from the P0 promoter (Fig. 7A). This effect was not due to reduced expression or instability of the mutant proteins, as Western blot analysis demonstrated a similar expression level of all used mutants and the wild-type protein (Fig. 7B). Unfortunately, the GFP-Sox10NES fusion protein exhibited only residual DNA binding affinity (Fig. 7C). Thus, we were unable to distinguish for this mutant whether reduced transactivation is attributed to inhibited nucleocytoplasmic transport or to reduced DNA-binding affinity. The GFP-Sox10L138A mutant, in contrast, showed a DNA-binding affinity similar to that of wild-type GFP-Sox10 as revealed by competition analysis with cold competitor DNA (Fig. 7D). For this mutant, reduced Sox10-dependent transactivation thus appears to be a consequence of impaired nucleocytoplasmic shuttling. To verify that the reduced transactivation capacity of the GFP-Sox10L138A mutant is indeed due to altered nucleocytoplasmic shuttling ability, we inserted the heterologous NES of human T-cell leukemia virus type 1 Rex between the GFP and the Sox10 part of the fusion protein. The resulting restoration of the nuclear export capacity resulted in a near complete retrieval of the transcriptional activity (Fig. 7A).
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Finally, we analyzed the effect of LMB on expression of endogenous target genes of Sox10 (31, 46). For this we used a stably transfected N2A Tet-On cell line capable of inducible expressing Sox10 (31). As shown by semiquantitative RT-PCR analyses, transcription of the two myelin genes P0 and PLP was strongly increased after doxycycline-dependent Sox10 induction (Fig. 7H). Additional LMB treatment prevented the Sox10-dependent increase of P0 and PLP transcription. GAPDH transcript levels were affected neither by treatment of doxycycline nor by treatment of doxycycline and LMB.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activity of transcriptional regulators can be modulated at various levels. They include phosphorylation, acetylation, assembly, and interactions with other proteins, including cofactors. The molecular mechanisms that regulate the activity of Sox proteins are just beginning to be studied. In the case of Sox9 phosphorylation by cyclic AMP-dependent protein kinase enhances the ability to transactivate the Col2a1 chondrocyte-specific enhancer (21). Furthermore, it was shown that Sox10 binds to DNA both as monomer and dimer with different functional consequences (32). In this study, we demonstrate that inhibition of the nuclear export activity of Sox10 leads to a decrease in its transactivation capacity, when measured both on transfected luciferase reporters and on endogenous target genes. Based on our findings, we propose that continuous shuttling and regular localization of Sox10 in the cytoplasm is essential for its optimal transactivation ability. At the moment we do not know why the cytoplasmic stay is important for the function of Sox10. One can speculate that Sox10 receives a posttranslational modification in the cytoplasm and is then transported into the nucleus to activate its target genes. After a given time in the nucleus the posttranslational modification is removed, activity is reduced, and Sox10 is consecutively exported into the cytoplasm to start the next cycle of shuttling. A similar model is proposed for STAT1 (25), in which tyrosine-phosphorylated STAT1 dimers that bind to DNA accumulate in the nucleus. Under this condition the NES is masked and not accessible for CRM1. Following dephosphorylation, STAT1 is released from the DNA, recognized by CRM1, and subsequently exported into the cytoplasm.
Sox10 is a key regulator during early phases of neural crest development (6). These cells contain high amounts of Sox10. In the peripheral nervous system, neural crest cells give rise to neurons and glia. While glia continue to depend on Sox10 for their development, neurons stop to express Sox10. The specific removal of Sox10 seems to be important for neuronal cell development. If Sox10 removal solely depends on down-regulation of expression of its gene, the process would be fairly slow. The balance of import and export provides an additional mechanism for the regulation of Sox10 activity. Changes in the kinetics of nucleocytoplasmic shuttling provide a rapid mechanism for inactivation of the existing Sox10 protein. Interestingly, Dutton et al. identified a Sox10 mutant in zebra fish, in which leucine 142 (equivalent to leucine 138 of the NES of human Sox10) is changed to glutamine (10). In contrast to the alanine substitution mutant analyzed in this paper, the glutamine substitution exhibits not only defective shuttling but also reduced DNA binding activity (data not shown). It is tempting to speculate that the neural crest defects observed in the zebra fish mutant are at least partially due to disrupted nucleocytoplasmic shuttling. Proof for the in vivo role of nucleocytoplasmic shuttling will have to await the generation of an animal model in which nuclear export of Sox10 is selectively abolished.
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ACKNOWLEDGMENTS |
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This work was supported by the DFG (grants We1326/7-2 and SFB473).
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FOOTNOTES |
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