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Vol. 16, Issue 6, 2660-2669, June 2005
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Institut de Génétique Humaine, Centre National de la Recherche Scientifique Unité Propre de Recherche 1142, 34396 Montpellier, France
Submitted December 10, 2004;
Revised March 7, 2005;
Accepted March 14, 2005
Monitoring Editor: Marianne Bronner-Fraser
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Phylogenetic analyses focusing on both HMG domain sequence and motifs outside led to the distinction of several subgroups, A to G (Bowles et al., 2000). Studies in several organisms, including Drosophila, Xenopus, chick, and mouse, have revealed that group B Sox genes (Sox1/2/3 in vertebrates and their homologues SoxNeuro and Dichaete in Drosophila) are all expressed in the developing central nervous system (CNS) from the earliest stages of neurogenesis onwards (for review, see Savare and Girard, 2002). These genes were shown to play fundamental roles in neural induction, maintenance of the neural phenotype, and maintenance of the identity of the neural precursors (Mizuseki et al., 1998; Bylund et al., 2003; Graham et al., 2003). In Drosophila, SoxNeuro (SoxN) expression in the neuroectoderm is regulated by the Dorsal and Dpp pathways (Crémazy et al., 2000; Stathopoulos et al., 2002). Loss of function mutations of SoxN are responsible for severe hypoplasia in the embryonic CNS, resulting from a drastic loss of neuroblasts in the lateral and intermediate regions of the neuroectoderm (Buescher et al., 2002; Overton et al., 2002). The target genes regulated by these Sox, and the molecular mechanisms that regulate their activity, are largely unknown.
The activity of transcription factors is regulated by several posttranslational modifications, including phosphorylation, acetylation, or ubiquitylation. Covalent modification by small ubiquitin like modifier (SUMO) has recently emerged as an important posttranslational modification involved in regulating several nuclear events, including transcription, nucleocytoplasmic shuttling, DNA replication and repair, chromosome dynamics, localization into discrete nuclear structures (for review, see Gill, 2003, 2004; Seeler and Dejean, 2003; Verger et al., 2003; Girdwood et al., 2004; Johnson, 2004). SUMO modification of transcription factors often has been associated with transcriptional repression, although the precise molecular mechanisms underlying this regulation are still a matter of debate (Gill, 2004; Girdwood et al., 2004). SUMOs are highly conserved in all eukaryotes. Whereas invertebrates contain only one SUMO, known as Smt3, four types of SUMO exist in vertebrates, SUMO1/2/3 and a recently identified SUMO4. SUMO modification (SUMOylation) consists in the covalent attachment of SUMO to a lysine residue in the protein substrate, at a consensus site 
KXE (where is a hydrophobic residue, either isoleucine, valine, or leucine) (Rodriguez et al., 2001; Sampson et al., 2001). Two enzymatic activities are involved in SUMO attachment: an E1 activating enzyme and an E2 conjugating enzyme known as Ubc9. A third class of enzymes was further identified, E3, that is not absolutely required for SUMO attachment in vitro but that might enhance specificity to the substrate in vivo. Several classes of SUMO E3 were discovered, including members of the PIAS family, RanBP2, and the polycomb group protein Pc2 (Sachdev et al., 2001; Kagey et al., 2003; Pichler et al., 2004). These enzymes were shown to enhance SUMO attachment in vitro and to target the substrate protein to particular nuclear compartments in vivo, such as promyelocytic leukemia (PML) bodies, nuclear pore, or polycomb bodies. SUMO modification is a reversible process, catalyzed by SUMO cleaving enzymes of the Ulp family (for recent review, see Johnson, 2004).
How Sox factor transcriptional activity is regulated still remains poorly understood. Sox often pair off with specific partners, leading to synergistic and context-dependent transcriptional regulation (for review, see Kamachi et al., 2000). In Sox9, several posttranslational events were shown to be essential for its activity, including protein kinase A-dependent phosphorylation (Huang et al., 2000), SUMO modification (Komatsu et al., 2004), and regulation of nucleocytoplasmic shuffling (Gasca et al., 2002). In this report, we present evidence that SoxNeuro is SUMO modified, both in Drosophila S2 cells and in HeLa cells. Lysine acceptor site maps within an inhibitory domain surrounded by two adjacent transactivation domains. SUMO modification of SoxN is associated with transcriptional repression. Our data also show that Sox3, the human counterpart of SoxN, is similarly regulated by SUMO modification. Finally, overexpression in Drosophila embryos of a SoxN form in which the SUMO acceptor lysine was mutated to arginine perturbs CNS development, strongly suggesting that the regulation of SoxN activity through SUMO modification is essential for CNS development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Lysine 439 and 375 were mutated to arginine in pGlomyc-SoxN and psG424-Sox3, respectively, by using PCR-based site-directed mutagenesis (Stratagene, La-Jolla, CA), and confirmed by sequencing. SoxN and human Sox3, and deletion mutants, were cloned in frame with GAL4 DNA binding domain in pSG424 (kindly provided by P. de Santa Barbara, Institut de Génétique Humaine, Montpellier, France), by using KpnI-XbaI PCR fragments. Details of the primers used are available upon request. pMK26-FlagHA-SoxN wild-type and K439R, and pUAS-SoxNK439R, were constructed as follows. An EcoRI fragment from pGlomyc-SoxN (wild-type and K439R) was first cloned in pcDNA-FlagHA (kindly provided by F. Poulat, Institut de Génétique Humaine, Montpellier, France) EcoRI-digested. The FlagHA-SoxN cassette was then removed by HindIII/NotI digestion, filled in with Klenow, and cloned in pMK26 (Act5C promoter) (kindly provided by A. Pélisson, Institut de Génétique Humaine, Montpellier, France) EcoRV-digested, and in pUAS. pcDNA-6His-SUMO2 and pcDNA-Ubc9 were provided by R. Hay (University of St. Andrews, St. Andrews, Scotland, United Kingdom). pSG5–6His-SUMO1 was provided by A. Dejean (Institut Pasteur, Paris, France). pPac-Smt3 and pPac-Ubc9 were provided by A. Courey (University of California, Los Angeles, CA). pcDNA-DN Ubc9 (C93S substitution) was provided by A. Sharrocks (University of Manchester, Manchester, United Kingdom).
Antibodies
Mouse anti-myc (Tebu, Le Perray en Yvelines, France), mouse anti-FLAG (Sigma-Aldrich, St. Louis, MO), mouse anti-hemagglutinin (HA) (Roche Di-agnostics, Indianapolis, IN), horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse (Amersham Biosciences, Piscataway, NJ), mouse anti-GAL4 DBD (Ozyme, St. Quentin en Yvelines, France), goat anti-Sox3 (Tebu), anti-mouse and anti-goat Alexa Fluor 568 (Molecular Probes, Eugene, OR), Cy3-conjugated mouse anti-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA), monoclonal anti-Fasciclin II (FasII) (Developmental Studies Hybridoma Bank, Iowa City, IA), and biotinylated goat anti-mouse and anti-rabbit (Amersham Biosciences) were all purchased commercially and used according to the manufacturer's instructions. Affinity-purified rabbit anti-SoxN was described previously (Crémazy et al., 2001). In Figure 6, anti-FasII and SoxN primary antibodies were detected with biotinylated secondary antibody and the ABC Elite kit (Vectastain; Vector Laboratories, Burlingame, CA). Whole mount embryo immunostainings were performed on formaldehyde-fixed Drosophila embryos by using standard protocols (Ashburner, 1989).
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For immunostainings, HeLa cells were fixed 18–24 h after transfection in 3.7% formaldehyde for 5 min followed by permeabilization in phosphate-buffered saline (PBS), 0.2% Triton for 10 min. S2 cells were plated on glass coverslips 48 h after transfection. Twenty-four hours later, cells were fixed in the same conditions. Cells were preincubated with PBS containing 1% bovine serum albumin and processed for immunostaining with either anti-myc or anti-FLAG, in HeLa and S2 cells, respectively, and Alexa Fluor 568 goat anti-mouse. The procedure was identical for HeLa cells transfected with pSG424-Sox3 derivatives, which were detected with goat anti Sox3/Alexa Fluor 568 anti-goat.
For luciferase assays, HeLa cells were transfected with pG5-Luc (reporter plasmid containing five multimerized Gal4 binding sites upstream of luciferase) (Promega, Madison, WI), pRL-SV40 (Promega) as internal control, and pSG424 effector plasmids. Luciferase activities were monitored 18–24 h after transfection using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. The firefly luciferase activities were normalized with Renilla luciferase activities from pRL-SV40.
SUMOylation Assays
HeLa cells were transfected with various combinations of plasmids (1.5 µg each, unless specified in the text, for a total amount of 7.5 µg of DNA), as indicated in figure legends. 6His-tagged SUMO-conjugated products were isolated and analyzed as follows. Eighteen to 24 h after transfection, cells were washed twice in PBS and lysed in 500 µl of lysis buffer (6 M guanidium chloride, 100 mM Na2HPO4, pH 8, 10 mM imidazole, and protease inhibitor cocktail). Extracts were then sonicated (8 pulses, 75 W), centrifugated, and the supernatant was subjected to nickel-agarose precipitation (QIAGEN) for 4 h at 4°C. After extensive washes (2 washes with 1 ml of lysis buffer, 2 washes with 1 ml of lysis buffer diluted 5 times in 50 mM Tris, pH 7.5 and containing 20 mM imidazole, and 2 washes with 1 ml 50 mM Tris, pH 7.5, containing 20 mM imidazole), samples were recovered in Laemmli buffer supplemented with 200 mM imidazole. Protein samples were resolved by SDS-PAGE and immunoblotted with anti-myc. For human Sox3, SUMOylation assay was conducted as described for SoxN, except that samples were immunoblotted with anti-GAL4 DBD to detect GAL4 DBD-Sox3 fusion proteins.
SUMOylation in Drosophila S2 cells was analyzed by immunoprecipitation. Cells were transfected with wild-type or K439R FLAG-HA–tagged SoxN and components of the SUMO machinery (Smt3 and Ubc9). Cells were lysed in lysis buffer (120 mM NaCl, 50 mM Tris, pH 8, 5 mM EDTA, 0.5% NP-40, 1 mM dithiothreitol, and protease inhibitors) containing 50 mM N-ethyl maleimide and incubated overnight at 4°C with anti-FLAG M2 affinity gel (Sigma-Aldrich). After two washes in lysis buffer and one in washing buffer (300 mM NaCl, 50 mM Tris, pH 7.5, 0.1% SDS, and 0.1% sodium deoxycholate), samples were resuspended in Laemmli buffer, resolved by SDS-PAGE, and immunoblotted with anti-HA to detect SoxN.
Fly Stocks
All Drosophila stocks were maintained on standard yeast-cornmeal medium at 25°C. Oregon R was used as wild-type stock. The following fly lines were used: pUAS SoxN (Blanco et al., 2005), da-GAL4 (Bloomington Stock Center, Indianapolis, IN), and ey-GAL4 (a gift of W. Gehring, Biozentrum, Basel, Switzerland). The pUAS-SoxN K439R stock was obtained by P element-mediated germline transformation by using standard protocols (Karess, 1985).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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KXE motif in substrate proteins, in which lysine is targeted for SUMOylation, was shown to be the binding site for the Ubc9 E2 ligase (Rodriguez et al., 2001; Sampson et al., 2001). The SUMO pathway is functionally conserved in vertebrates and invertebrates. Indeed, it was shown that the Drosophila dSmt3 conjugation system could efficiently recognize and modify human SUMO1 substrates (Sapetschnig et al., 2002). Thus, because SoxN sequence contains a consensus site for SUMO modification, IKSE, in which lysine 439 could be potentially targeted, we used HeLa cells as a paradigm to analyze SoxN SUMOylation. Our assay consisted in transfecting cells with myc-tagged SoxN, together with6His-tagged SUMO1 or SUMO2. Cell extracts were prepared under denaturing conditions to inhibit SUMO protease activities, and incubated with nickel-nitrilotriacetic acid (Ni-NTA) resin, to specifically bind the 6His-tagged, SUMO-modified proteins. Samples were then probed by Western blot for the presence of myc-SoxN. Because SoxN contains in its sequence a stretch of five histidines (amino acid position 58–62), it was able to bind efficiently to the resin when myc-SoxN plasmid was transfected alone (Figure 1A). Cotransfecting 6His-SUMO1 or 6His-SUMO2 resulted in the purification of a slower migrating, SUMO-modified, SoxN form. The stoichiometry of this SUMO-modified SoxN form was directly proportional to the quantity of SUMO (1 and 2) plasmid transfected. In SUMO1, because it was apparently less expressed, it was necessary to increase the quantity of transfected 6His-SUMO1 plasmid to detect SUMO-modified SoxN (Figure 1A, right). Cotransfecting Ubc9, the SUMO E2 ligase, did not significantly increase the ratio of SUMO-modified SoxN, likely because HeLa cells contains sufficient amounts of endogenous Ubc9 (Figure 1B). Finally, we use a dominant negative form of Ubc9 (DN Ubc9), able to interfere with the endogenous SUMO pathway (Yang et al., 2003) and analyzed SoxN SUMOylation. We observed that cotransfecting increasing quantities of DN-Ubc9 together with SoxN and 6His-SUMO2 resulted in a significant decrease in SUMO modification of SoxN (Figure 1C). Collectively, these data demonstrate that SoxN is efficiently SUMO modified in transfected HeLa cells, both with SUMO1 and SUMO2.
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Lysine 439 in the IKSE SUMO consensus site was mutated to arginine, and tested for SUMO modification using the same assay. As shown in Figure 1D, no SUMO modified SoxN form was detected when K439R mutant was transfected in HeLa cells in the presence of 6His-SUMO2. The same result was obtained when SUMO1 was used instead of SUMO2, and when Ubc9 was cotransfected together with SUMO1 or SUMO2 (our unpublished data). This result showed that Lysine 439 within the IKSE motif is the major SUMO acceptor site in SoxN.
We next analyzed whether SoxN was also SUMO modified in cultured Drosophila S2 cells. Reverse transcription-PCR and immunostaining experiments revealed that these cells contained no detectable levels of SoxN RNA and protein. The SUMOylation assay consisted in transfecting FLAG-HA tagged SoxN, together with components of the SUMO machinery (Smt3 and Drosophila Ubc9). Cell lysates were immunoprecipitated with anti-FLAG, and immunoblotted with anti-HA to detect SoxN. A SUMO-modified SoxN form was detected at the expected molecular size when wild-type SoxN was overexpressed in S2 cells, and this form was absent when K439R mutant was transfected (Figure 1E). This experiment shows that SoxN is SUMO modified in Drosophila and confirmed that K439 is the major SUMO acceptor site. The SUMO-modified SoxN form was detected in extracts prepared from S2 cells transfected with FLAG-HA-SoxN alone (Figure 1E). This suggested that even when the components of the SUMO machinery were not overexpressed, SoxN was endogenously SUMO modified and that SoxN SUMO modification does not simply result from Smt3/Ubc9 overexpression.
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The C-terminal region was further deleted, and the corresponding constructs were monitored for their transcriptional activity (Figure 2B). Deleting the region between the HMG box and the SUMOylation site (constructs 321–573 and 425–573) significantly reduced the transcriptional activity. Deleting the C-terminal domain up to the SUMOylation site (constructs 256–499 and 256–469) resulted in a similar loss of transcriptional activation. This suggests that SoxN contains two regions important for transcriptional activation, both containing alanine repeats. Constructs 256–469, 256–434, 425–573 and 442–573 were used to study the importance of the SUMOylation site in SoxN transcriptional activity. We observed that the removal of the IKSE motif in these constructs led to a dramatic increase in transcriptional activity. Indeed, whereas constructs 256–469 and 425–573 exhibited, respectively, luciferase activity 1.1- and 2.3-fold higher than the control, the same constructs deleted of the SUMO motif displayed luciferase activity 42- and 22.5-fold higher than the control, respectively. These results show that the region 434–442 containing the IKSE motif exerts an inhibitory effect in cis on the two adjacent transcriptional activation domains (TADs), localized at positions 256–434 and 442–573.
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We also examined SoxN transcriptional activity when DNUbc9 was cotransfected to diminish endogenous SUMO machinery activity. We observed a dose-dependent increase in SoxN transcriptional activity, with relative luciferase activity ranging from 1 when SoxN was transfected alone to 5.6 when DNUbc9 was coexpressed (Figure 3B). Hence, DNUbc9 was effective in inhibiting SUMO modification of SoxN (Figure 1C), which resulted in increased SoxN activity (Figure 3B). By contrast, we found that cotransfecting DNUbc9 up to 2 µg had little or no effect on the luciferase activity of the GAL4 DBD alone (Figure 4D). Together, these two experiments show that SoxN SUMO modification represses its transcriptional activity.
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; Bowles et al., 2000). Closer examination of their sequence revealed that all three proteins contain a KXE motif located in their C-terminal region (Figure 4A). In these three proteins, C-terminal region was shown to contain sequences necessary for transcriptional activity (Bowles et al., 2000, and references therein). We used SUMOylation assay in HeLa cells to determine whether human Sox3 was SUMO modified. Cotransfecting 6His-SUMO1 (Figure 4C, left) or 6His-SUMO2 (right) together with GAL4 DBD-Sox3 C-ter resulted in the purification of a slowly migrating SUMO-modified band, which was not detected when lysine 375 in the putative SUMO acceptor site was mutated to arginine. As already observed for SoxN, DN Ubc9 when cotranfected in the cells is able to significantly impair Sox3 SUMO modification (Figure 4C). This demonstrated that lysine 375 is the major SUMO acceptor site in human Sox3.
As shown previously, SoxN contains two TADs, in which long stretches of alanine repeats are present (n = 12 in TAD1 and n = 14 in TAD2). Similarly, human Sox3 contain four alanine repeats (n = 15, 7, 6, and 10), all being localized N-terminally to the VKSE SUMOylation motif (Figure 4B). We monitored luciferase activity of GAL4 DBD-Sox3 216–447 derivative, in the absence or presence of DNUbc9 (Figure 4D). Sox3 displayed a moderate transcriptional activity (2-fold over the GAL4 DBD alone), which was increased when the endogenous SUMO machinery was challenged with DN Ubc9 cotransfection (4-fold over the GAL4 DBD alone). By contrast, the activity of the GAL4 DBD was similar in the absence or presence of DN Ubc9. Finally, the activity of the K375R Sox3 mutant was compared with its wild-type counterpart (Figure 4E). As already observed for SoxN, the single K-to-R mutation in the SUMO acceptor site resulted in a significant increase in transcriptional activity (23-fold higher than the GAL4 DBD alone, and 9-fold higher than its wild-type counterpart). Collectively, these data show that human Sox3 is SUMO modified on lysine 375 in HeLa cells and that Sox3 SUMO modification is associated with transcriptional repression.
SUMO Modification Is Not Associated with Changes in SoxN and Sox3 Cellular Localization
SUMO modification has been shown to regulate subcellular localization of several targets, including transcription factors, although it does not seem to be a general mechanism by which SUMO affects transcription. For example, SUMO was shown to be involved in nuclear import/export, targeting to the nuclear pore and to discrete nuclear compartments such as PML bodies (for review, see Seeler and Dejean, 2003; Gill, 2004). In transfected HeLa cells, myc-tagged SoxN is found exclusively in the nucleus, localized at many punctuate sites, and with higher staining found at the nuclear membrane (Figure 5) We found that this nuclear pattern was not modified in the following conditions: when SUMO acceptor lysine was mutated to arginine, when SUMO1/2 were cotransfected with SoxN, or when the endogenous SUMO pathways was challenged with DNUbc9 transfection (Figure 5). Similar conclusions were reached when comparing SoxN nuclear distribution in Drosophila S2 cells with its K439R counterpart or when cotransfecting Smt3 (Figure 5). Finally, we monitored the distribution of GAL4 DBD-Sox3 C-ter wild-type and K375R derivatives with anti-Sox3 antibody and observed similar nuclear distribution for the wild-type and the SUMO-deficient K375R form (Figure 5). Collectively, these data implies that SUMO modification of SoxN and Sox3 is not associated with major redistribution of the proteins to specific subnuclear compartments in transfected cells.
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Overexpression of SoxN K439R Perturbs Drosophila CNS Development
To analyze the impact of SUMOylation on SoxN function in vivo, we compared the phenotypic consequences of overexpressing either wild-type SoxN or the K439R SUMO deficient form during Drosophila development. For this purpose, we used the UAS-GAL4 system to drive the expression of both proteins in larval imaginal discs. Although overexpressing wild-type SoxN results in severe defects in the formation of adult structures (including defects in wings, thorax, and legs), it was impossible to obtain adults from the SoxN K439R-overexpressing larvae, because this overexpression was lethal during larval and/or early pupal periods (our unpublished data). When SoxN K439R expression was driven in the eye imaginal discs by using ey-GAL4, it was nevertheless possible to collect few adult escapers from the pupal case, showing a headless phenotype (Figure 6A). By contrast, overexpression of wild-type SoxN with ey-GAL4 led to a much milder phenotype, with reduction or complete absence of the eye structures (Figure 6, B and C). These phenotypes could result from interference of SoxN ectopic expression with the development pathway initiated by eyeless in the eye imaginal discs, a phenomenon already observed when driving the expression of several transcription factors in the developing eye and called developmental pathway interference (Jiao et al., 2001). These results suggested that wild-type and SUMO-deficient SoxN behave differently when overexpressed in Drosophila and prompted us to examine the effects of their overexpression on embryonic CNS development. For this purpose, the da-GAL4 driver line was used, which results in ubiquitous expression starting at embryonic stage 8. Ubiquitous K439R SoxN expression resulted in lethality at the end of embryogenesis, with severe defects in the CNS as observed with HRP (Figure 6, D–F) and Fasciclin II stainings (Figure 6, G and H). Indeed, defects included fusion or absence of commissures (Figure 6F), reduction or absence of longitudinal axon tracts (Figure 6F) and strong perturbation of the regular axonal fasciculation pattern as seen with FasII staining (Figure 6H). In marked contrast, overexpressing the wild-type SoxN form had no detectable effect on embryonic CNS development (Figure 6, E and G). Immunostaining with anti-SoxN antibodies in these embryos revealed that both proteins were expressed at similar levels, and both localized in the nuclei (Figure 6, I–K). Collectively, these data show that SUMO modification is important for regulating the activity of SoxN in vivo and strongly suggest that SUMO modification of SoxN is involved during CNS development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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KXE motif) in all mammalian and Drosophila Sox proteins (Table 1). One or several KXE motifs are present in some but not all Sox genes, these motifs being usually conserved within a given subgroup between Drosophila and humans. These include group B1 (H.s Sox1/2/3 and D.m SoxN), group C (H.s Sox11 and D.m SoxC), group D (H.s Sox5/6/13), group E (H.s Sox8/9/10 and D.m Sox100B), group F (H.s Sox17), and group H (H.s Sox30). Recently, Sox9 was shown to be SUMO modified, and SUMO modification was associated with transcriptional repression (Komatsu et al., 2004). In all the other groups (B2, F, and G), no KXE motif is present (except Drosophila SoxB2–2, human group C Sox11 and group F Sox17), suggesting that these proteins are not SUMO modified. To confirm this, we used the same SUMOylation assay as described in this report for SoxN and Sox3, and were unable to detect SUMO modified human Sox7, mouse Sox15 and Drosophila Dichaete (respectively, group F, G, and B) (our unpublished data). Thus, based on our data and that of Komatsu et al. (2004) and the presence of KXE motif in various Sox, one can postulate that SUMO modification might be used to regulate several Sox group genes.
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Our results show that SUMO modification of the CNS-specific group B1 SoxN and Sox3 proteins was conserved during evolution to regulate their transcriptional capacity. Based on the presence of KXE motif in group B1 proteins (SoxN in Drosophila and Sox1/2/3 in humans), and its absence in group B2 (Dichaete in Drosophila and Sox14/21 in humans), it is tempting to speculate that these two subgroups differ in their ability to be regulated by SUMOylation. This is particularly interesting because in Drosophila, SoxN and Dichaete were shown to partially overlap in their expression and function within the neuroectoderm, suggesting that these genes are to some extent functionally redundant in the developing CNS but that there must exist molecular mechanisms responsible for their specificity of action in restricted areas of the CNS (interactions with specific partners? posttranslational modifications?) (Buescher et al., 2002; Overton et al., 2002; Gomez-Skarmeta et al., 2003). Furthermore, it was shown in chick that group B2 Sox14/21 could bind and differentially regulate 
1-crystallin gene regulatory sequences, known to be regulated by group B1 Sox1/2/3 factors in vivo (Uchikawa et al., 1999). These observations suggested that target of group B genes might be regulated by the counterbalance of activating and repressing Sox proteins in restricted sites of the developing CNS. In light of our results, SUMOylation might be one of the mechanisms used for this purpose.
As shown here, substitution of lysine 439 to arginine within SoxN IKSE motif impaired SoxN SUMO modification in both transfected HeLa and S2 cells. SoxN transcriptional activity was dramatically enhanced in three conditions: in the substitution mutant K439R, in the deletion mutants where the IKSE motif was deleted, and when the dominant negative form of Ubc9 was used to interfere with the endogenous SUMO machinery. This correlation between transcriptional repression and the ability of SoxN to be SUMOylated strongly suggests that SUMO conjugation to SoxN results in transcriptional repression. Similar results were obtained for its human counterpart Sox3. Many of the SUMO-modified proteins identified to date are transcription factors, and in most cases, SUMO modification has been associated with transcriptional repression. Nevertheless, the molecular mechanisms underlying this repression are still a matter of debate. In some cases, SUMO modification was associated with the relocalization of the targeted factor to specialized repressive subnuclear structures such as PML bodies (for review, see Gill, 2003, 2004; Girdwood et al., 2004). In SoxN and Sox3, our data in HeLa and S2 cells suggest that SUMOylation is apparently not associated with major changes in the nuclear localization of these proteins (Figure 5). This was also evident in vivo, because the wild-type and K439R SoxN forms both localized similarly in the nuclei (Figure 6).
In both SoxN and Sox3, we found that the KXE motif is targeted for SUMOylation, and constitutes an inhibitory domain able to affect the activity of adjacent TADs. Interestingly, this motif is surrounded by conserved proline residues, reminiscent of the SC synergy domain (consensus P-X0-4-KXE-X0-3-P) found in several transcription factors, including SP3, c-myb, C/EBP, and Sox9 (Komatsu et al., 2004, and references therein). Potential SC motifs also are found in other Sox: H.s Sox6, H.s Sox8, and H.s Sox30 (Table 1). SC motif is both necessary and sufficient to limit transcriptional synergy, because its disruption selectively enhances synergistic activation at compound response elements without altering the activity driven from a single site (Iniguez-Lluhi and Pearce, 2000). Thus, SUMOylation of the SC domain is believed to modulate higher order interactions among transcriptional regulators. This motif in Sox proteins might behave as SC domain, because these factors are known to pair off with specific partners to exert full and synergistic activity in a context dependent manner (Kamachi et al., 2000). Because SUMO modification is believed to modulate protein–protein interactions, it will be of interest to examine whether Sox SUMOylation is able to interfere with their ability to interact with their partners.
Using transgenic Drosophila lines, we obtained strong evidence that SUMOylation regulates the activity of SoxN in vivo. Indeed, overexpressing the SUMO-deficient K439R SoxN form resulted in strong defects in embryonic CNS. Because the GAL4 driver used for embryonic overexpression is ubiquitous, we interpret these results as the capacity of the nonSUMOylable form to interfere with endogenous SoxN in the cells were SoxN is expressed (neuroblasts and neurons). In addition, our experiments where the wild-type and K439R SoxN proteins were overexpressed in larvae clearly showed that the two forms display different activity in vivo, further demonstrating the functional relevance of SoxN SUMOylation in vivo. Because the K439R form is a strong transcriptional activator as observed in our luciferase assays in transfected cells, we can postulate that the repressing activity of SoxN is important for the proper development of embryonic CNS. Further work will be required to demonstrate whether SUMOylation regulates SoxN activity in all the different cell types where the protein is expressed (embryonic, larval and adult CNS, larval and adult eyes, and larval leg imaginal discs).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: DBD, DNA binding domain; HMG, high mobility group; Sox, sry HMG box; SoxN, SoxNeuro; SUMO, small ubiquitin-like modifier; TAD, transactivation domain.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Franck Girard (fgirard{at}igh.cnrs.fr).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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