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• W2008209085 abstract "Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes. Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes. high mobility group acid-rich β-galactosidase chloramphenicol acetyltransferase cytomegalovirus δ-crystallin enhancer enhanced chemifluorescence electrophoretic gel mobility shift analysis fibroblast growth factor 4 Pit-Oct-Unc transactivation domain The Sox family of transcription factors is comprised of a diverse group of proteins whose pattern of expression is regulated both spatially and temporally (1Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (752) Google Scholar). Sox factors are related by the homology (usually >50%) found within their high mobility group (HMG)1 DNA binding domains. Sox HMG domains have been found to bind to the consensus sequence 5′-(A/T)(A/T)CAA(A/T)G-3′ in the minor groove of DNA (2Harley V.R. Lovell-Badge R. Goodfellow P.N. Nucleic Acids Res. 1994; 22: 1500-1501Crossref PubMed Scopus (333) Google Scholar). As the number of Sox proteins has grown to more than 20 members in multiple species, including both vertebrates and invertebrates, this family has been divided into seven subgroups, the members of which contain high homology within both the HMG domain and flanking regions (1Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (752) Google Scholar). In some cases, subgroup members have similar patterns of expression during development and perhaps redundant functions. For example, the structurally similar group B members Sox-1, -2, and -3 show overlapping expression in the fetal nervous system, and it has been suggested that they perform similar functions (3Collignon J. Sockanathan S. Hacker A. Cohen-Tannoudji M. Norris D. Rastan S. Stevanovic M. Goodfellow P.N. Lovell-Badge R. Development. 1996; 122: 509-520PubMed Google Scholar, 4Kamachi Y. Uchikawa M. Collignon J. Lovell-Badge R. Kondoh H. Development. 1998; 125: 2521-2532Crossref PubMed Google Scholar). Additionally, the group C members Sox-4 and Sox-11 show high homology within their HMG domain and C-terminal tail, and they may also have complementary roles in the developing nervous system (5Cheung M. Abu-Elmagd M. Clevers H. Scotting P.J. Brain Res. Mol. Brain Res. 2000; 79: 180-191Crossref PubMed Scopus (147) Google Scholar). The observation that the HMG DNA binding domains of many members of the Sox family bind a consensus sequence has prompted the question of whether there are selective mechanisms in place to recruit a specific Sox protein to a gene regulated by an HMG binding site. A review of the literature reveals that genes rely on a diverse array of mechanisms to regulate DNA binding among members of other transcription factor families, which may also apply to Sox proteins. A particularly intriguing example of DNA binding regulation has been found within multiple members of the Ets family. First characterized in Ets-1, the affinity of these proteins for DNA is tightly regulated by autoinhibitory regions flanking the DNA binding domain (6Hagman J. Grosschedl R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8889-8893Crossref PubMed Scopus (132) Google Scholar, 7Petersen J.M. Skalicky J.J. Donaldson L.W. McIntosh L.P. Alber T. Graves B.J. Science. 1995; 269: 1866-1869Crossref PubMed Scopus (174) Google Scholar). Interestingly, the ubiquitous HMG-1 and HMG-2 proteins employ a similar regulatory strategy whereby their highly acidic C-terminal tails are capable of negatively affecting the ability of those proteins to bind DNA (8Isackson P.J. Beaudoin J. Hermodson M.A. Reeck G.R. Biochim. Biophys. Acta. 1983; 748: 436-443Crossref PubMed Scopus (10) Google Scholar). Importantly, although the autoinhibitory domains of both Ets and HMG proteins repress DNA binding, they can be neutralized by post-translational modification or by interaction with partner proteins, which allows their recruitment to be exquisitely regulated in a gene-specific manner (7Petersen J.M. Skalicky J.J. Donaldson L.W. McIntosh L.P. Alber T. Graves B.J. Science. 1995; 269: 1866-1869Crossref PubMed Scopus (174) Google Scholar, 9Goetz T.L. Gu T.L. Speck N.A. Graves B.J. Mol. Cell. Biol. 2000; 20: 81-90Crossref PubMed Scopus (116) Google Scholar, 10Cowley D.O. Graves B.J. Genes Dev. 2000; 14: 366-376PubMed Google Scholar, 11Das D. Scovell W.M. J. Biol. Chem. 2001; 276: 32597-32605Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 12Stemmer C. Schwander A. Bauw G. Fojan P. Grasser K.D. J. Biol. Chem. 2002; 277: 1092-1098Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although specific evidence of DNA binding autoregulation has not yet been observed in Sox proteins, it is clear that cis-regulatory elements are capable of selecting specific Sox transcription factors. Attempts to uncover the mechanisms through which Sox specificity is achieved have revealed that recruitment often requires proper interaction with other transcription factors at adjacent binding sites on the DNA. This is illustrated by one of the cis-regulatory elements that control expression of the fibroblast growth factor 4 (FGF-4) gene in embryonal carcinoma cells. FGF-4 expression depends on a distal enhancer (13Curatola A.M. Basilico C. Mol. Cell. Biol. 1990; 10: 2475-2484Crossref PubMed Scopus (78) Google Scholar, 14Ma Y.G. Rosfjord E. Huebert C. Wilder P. Tiesman J. Kelly D. Rizzino A. Dev. Biol. 1992; 154: 45-54Crossref PubMed Scopus (69) Google Scholar), which contains an essential binding site for Sox-2 (15Yuan H. Corbi N. Basilico C. Dailey L. Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (643) Google Scholar, 16Johnson L.R. Lamb K.A. Gao Q. Nowling T.K. Rizzino A. Mol. Reprod. Dev. 1998; 50: 377-386Crossref PubMed Scopus (25) Google Scholar, 17Nowling T. Bernadt C. Johnson L. Desler M. Rizzino A. J. Biol. Chem. 2002; (December 17, 10.1074/jbc.M207567200)Google Scholar). However, Sox-2 does not appear to be capable of substantial activation of this gene on its own, but rather requires cooperation with the POU transcription factor Oct-3 (18Ambrosetti D.C. Basilico C. Dailey L. Mol. Cell. Biol. 1997; 17: 6321-6329Crossref PubMed Scopus (306) Google Scholar, 19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The presence of Oct-3 at a site adjacent to Sox-2 promotes cooperative DNA binding, resulting in a synergistic increase in transactivation (18Ambrosetti D.C. Basilico C. Dailey L. Mol. Cell. Biol. 1997; 17: 6321-6329Crossref PubMed Scopus (306) Google Scholar,19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The partnership of Sox-2 and Oct-3 is extremely specific for these two proteins because it requires stereospecific alignment and protein-protein interaction between the HMG and POU DNA binding domains when Sox-2 and Oct-3 are present at their adjacent binding sites (18Ambrosetti D.C. Basilico C. Dailey L. Mol. Cell. Biol. 1997; 17: 6321-6329Crossref PubMed Scopus (306) Google Scholar). Additionally, the role of Oct-3 cannot be fulfilled by Oct-1 for theFGF-4 enhancer, indicating that only certain Sox-POU combinations can lead to productive regulation of a particular gene (15Yuan H. Corbi N. Basilico C. Dailey L. Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (643) Google Scholar). Specific partnerships have also been observed among other members of the Sox and POU families. Utilizing the binding sequence found in the FGF-4 enhancer, it was determined that Sox-10 specifically partners with Oct-6, and Sox-11 cooperates optimally with the POU factors Brn-1 and Brn-2 (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 21Kuhlbrodt K. Herbarth B. Sock E. Hermans-Borgmeyer I. Wegner M. J. Neurosci. 1998; 18: 237-250Crossref PubMed Google Scholar). These observations have led to the hypothesis that a Sox-POU combinatorial code may exist as a mechanism of transcriptional regulation (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Although significant progress has been made in understanding the differential function of Sox proteins, both on their own and in combination with other factors, little is known about the mechanisms involved. To elucidate the mechanisms through which Sox proteins may be regulated, we initiated a functional comparison of Sox-2 and Sox-11 using promoter/reporter gene constructs that contain HMG and POU binding sites. Through this study, we determined that Sox-2 performs very differently than Sox-11, being a much weaker transactivator, while at the same time binding more strongly to DNA in vitro. To understand the mechanisms involved in the functional differences between Sox-2 and Sox-11, we addressed three questions. First, what role does each of the domains of Sox-2 and Sox-11 play in differential gene activation? Second, how specific are Sox-2 and Sox-11 partnerships with POU proteins, and which regions of Sox-2 and Sox-11 are important for Sox-POU partnerships? Third, which domain(s) are critical for the decreased DNA binding of Sox-11 compared with Sox-2? We have addressed these questions using Sox deletion mutants and Sox-2/11 chimeric proteins created by interchanging the N terminus, HMG DNA binding domain, or C terminus of both proteins. These studies led to the discovery that the C-terminal transactivation domains play the primary role in the differential capability of Sox-2 and Sox-11 to transactivate. Additionally, we determined that Sox-11, but not Sox-2, can cooperate with Brn-2 and that the Sox-11/Brn-2-specific cooperation requires both adjacent binding of the two factors to DNA and the HMG domain of Sox-11. Therefore, this study further supports the hypothesis that a Sox-POU combinatorial code exists (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) and suggests that this interaction code is dependent on the HMG domain of the Sox protein involved. Finally, we also identified a novel domain within a conserved region of Sox-11 which is capable of negatively regulating both its ability to bind DNA in vitro and activate gene expressionin vivo. Together, this work significantly enhances our understanding of the mechanisms that influence selective recruitment of Sox proteins through partner specificity and DNA binding autoregulation. Constructs pCATSO3, pCMVSox-2F1–180, pCMVSox-2, pCMVOct-3, pCMVOct-1, and CMV-β-gal have been described previously (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The promoter/reporter construct pCATDC3 was constructed similarly to pCATSO3 as follows. Complementary oligonucleotides (synthesized by the Eppley Cancer Institute Molecular Biology Core Facility) containing two sets of the HMG site from the DC5 enhancer site and the POU binding sites (in boldface) from the FGF-4 enhancer region and BamHI sites at both ends (5′-GATCCTCATTGTTGTGATGCTAATGGCTCATTGTTGTGATGCTAATGGAG-3′) were annealed and ligated to generate multimers. Differences in the DC5 and FGF-4 HMG binding sequence are underlined. These multimers were then ligated into a BglII site upstream from the SV40 promoter of the pCAT vector (Promega) to generate pCATDC3, which contains a total of six tandem copies of the HMG and POU sites. Similarly, pCATS4 contains a total of four tandem copies of the FGF-4 HMG site and was made using identical primers as pCATSO3, with the exception of a scrambled POU site. The upper primer is as follows with the HMG and scrambled POU sites in bold: (5′-GATCTCTTTGTTTGGCGGATCATGGCTCTTTGTTTGGCGGATCATGGA-3′). The plasmids pCMVSox-11 and pCMVBrn-2 were gifts from Michael Wegner (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Sox-11F was constructed using the primers FLAGSox-11 (5′-CGTGCTGGTACCGCCACCATGGACTACAAGGACGACGATGATATGGTGCAGCAGGCCGAGAGC-3′) and Sox-11/395 (5′-GCTGGATCCGACCGCCACGACTGCCTCCCG-3′) to amplify the coding region from CMV5-Sox-11. This PCR incorporated the FLAG epitope sequence, preceded by a Kozak sequence and a KpnI site, onto the 5′-end of the coding region of Sox-11 and aBamHI site at the 3′-end. TheKpnI/BamHI sites were then digested and the Sox-11F sequence ligated into the KpnI/BamHI sites of the CMV5 expression vector. The Sox-2F expression plasmid referred to in this report was constructed by removing the Sox-2F sequence from the pCEP4 vector (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) using theKpnI/BamHI sites and religating the Sox-2F sequence into the same sites present in CMV5. Site-directed mutagenesis or deletions were carried out using a modification of the QuikChange (Stratagene, La Jolla, CA) method. All amino acid numbers are based on the wild-type Met being defined as 1, not the Met included with the FLAG tag. Briefly, two primers, complementary to the sequence flanking the targeted region and to each other, were used in PCR amplification of the entire plasmid, thus modifying the indicated template. Unless otherwise noted, each of the primers identified herein represents the sense strand of a perfectly complementary pair of primers used during PCR. To begin the construction of the Sox-2/11 chimeras, unique restriction sites were incorporated at the N-terminal and C-terminal flanks of the HMG box of both the Sox-2F and Sox-11F plasmid. Sites were chosen in such a way that little alteration would be made to the wild-type coding sequence. Mutagenesis to include a NheI site was performed with either the sox2mut1 (5′-GGCAACCAGAAGAATGCTAGCCCGGAACGTGTCAAGAGGCCC-3′) or sox11mut1 (5′-CCGGACTGGTGCAAGACGGCTAGCGGCCACATCAAACG-3′) primer pair. The NheI alteration added a Ser following Ala-39 of Sox-2F but did not affect the coding sequence of Sox-11F. The products of these PCR reactions were then used in a second round of mutagenesis to include a NotI site using either the sox2mut2 (5′-GTACACGCTTCCCGCGGCCGCTTTGCTCGCCCCCGG-3′) or sox11mut2 (5′-GCCAAGCCCAGCGCGGCCGCACAGAGCCCGGACAAGAGC-3′) primer pair. This replaced Gly-131 and Gly-132 of Sox-2F as well as Ala-134 and Gly-135 of Sox-11F with the amino acids Ala-Ala-Ala. This cloning resulted in Sox-2F double mutant and Sox-11F double mutant, each of which contained a KpnI and BamHI site at their extreme 5′- and 3′-ends, respectively, as well as an NheI andNotI site at the 5′- and 3′- respective ends of their HMG boxes. These sites made it possible to use standard restriction digests and ligations to interchange either the N-terminal, HMG, or C-terminal region of Sox-2F with the corresponding region of Sox-11F, resulting in the creation of the chimeric plasmids Sox-11-2-2F, -11-11-2F, -2-11-2F, -2-11-11F, -2-2-11F, and -11-2-11F. To create Sox-11FΔTAD, the primer pair sox11Δtad (5′-CTCTACTACAGCTTCAAGTGAGCGGCCGCAAACATCACCAAGCAGCAG-3′) was used to incorporate a stop codon following Lys-282 of Sox-11F. The Sox-11F and Sox-11FΔTAD expression constructs were then used as the templates to create Sox-11FΔAR and Sox-11FΔARΔTAD, respectively, using the primer pairs of sox11Δar (5′-GCCAAGGTGGTCTTCCTGGACGCGCAGCAGCAACCCCCTCAG-3′) to delete amino acids 190–223. To facilitate in vitro translation of the various Sox proteins, the coding regions from each of the above CMV5-derived vectors were cloned into theKpnI/BamHI sites of pCRScript (Stratagene), downstream from the T7 promoter. The identity of all plasmids modified by PCR-based mutagenesis as well as pCATS4 and pCATDC3 was verified by sequence analysis at the Eppley Cancer Institute Molecular Biology Core Facility. HeLa cells were maintained and transfected using the calcium phosphate precipitation method as described previously (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). All transfections were performed in triplicate with representative experiments shown. Plasmid DNA was isolated and purified using Qiagen tip-500 columns. HeLa cells were transfected with 5 μg of the relevant plasmids as described above, and extracts were prepared 2 days post-transfection. Whole cell protein extracts were prepared as described previously (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Nuclear extracts were prepared using the NE-PER kit (Pierce) following the manufacturer's protocol. The isolated nuclei from ∼6–7 × 106 cells were lysed in 100 μl of nuclear extraction reagent supplemented with various protease and phosphatase inhibitors (22Scholtz B. Lamb K. Rosfjord E. Kingsley M. Rizzino A. Dev. Biol. 1996; 173: 420-427Crossref PubMed Scopus (33) Google Scholar). When preparing Sox-11F, Sox-11FΔTAD, and Sox-11FΔARΔTAD for the nuclear extracts used in the electrophoretic gel mobility shift assay (EMSA) shown in Fig. 6c, calpain inhibitor I was added to the growth media 4 h before lysis and was included in the extraction buffer to minimize degradation caused by potential proteasome activity. Extracts were stored at −80 °C. Sox proteins produced through in vitro transcription and translation were made using the T7 Quick Coupled Rabbit Reticulocyte Lysate (Promega) transcription/translation system. Western blotting was performed using 10 μl of in vitrotranslated lysate or nuclear lysate from 5 × 105transfected HeLa cells as described (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) using the anti-FLAG M2 antibody (Sigma). Proteins were detected using the enhanced chemifluorescence (ECF) kit (Amersham Biosciences) and scanned on a Storm PhosphorImager (Molecular Dynamics). Quantitation was performed using the ImageQuant 5.0 analysis software (Molecular Dynamics). Western blots were performed in duplicate or triplicate with representative blots shown. Gel mobility shift analysis was based on the method of Fried and Crothers as modified by this laboratory (23Kelly D. Scholtz B. Orten D.J. Hinrichs S.H. Rizzino A. Mol. Reprod. Dev. 1995; 40: 135-145Crossref PubMed Scopus (28) Google Scholar). Complementary oligodeoxynucleotide probes were annealed for each probe or competitor, and the resulting double-stranded oligodeoxynucleotide probes were labeled with [α-32P]dCTP by Klenow fill-in reaction of the single-stranded regions at the end of each double-stranded oligodeoxynucleotide probe (lowercase bases). The wild-type Sox probe (hmg1) contained a single HMG binding site (bold), which was based on the sequence present in the FGF-4 enhancer, whereas in the mutant Sox probe (HMGmut) this sequence is scrambled (underlined). The sequence of hmg1 (sense strand) is 5′-tagaAAACTCTTTGTTTGCCATGTCG-3′, and the sequence of HMGmut (sense strand) is 5′-tagaAAATTAGTCGAATGCCATGTC-3′. The hmg2 probe was created to mimic the pCATSO3 reporter construct in that it contains multiple HMG binding sites separated by 14bp. The sequence of hmg2 (sense strand) is 5′-tagaTCTTTGTTTGGCGGATCATGGCTCTTTGTTTGGCGGATCATGGA-3′. For gel mobility shift and competition assays, 1–2 μl of nuclear lysate or 3–4 μl of in vitro translated protein was incubated (at room temperature) for 30 min in a 20-μl volume containing 20 mm HEPES pH 7.6, 1 mm EDTA, 2 mm MgCl2, 20% glycerol, 50 mmNaCl, 5 μg of bovine serum albumin, 5 μg of p(dGdC)p(dGdC), and 20,000 cpm of labeled probe (final concentration of 0.5–1 nm) with or without competitor. The exact volume of nuclear lysate used in each assay was determined by first normalizing for differences in Sox protein concentration based on Western blot analysis. In gel mobility supershift assays, reaction mixtures were incubated for 1 h at 4 °C with the indicated antibody before addition of the probe, after which the reaction was continued at room temperature for 30 min. Immediately after the completion of binding, the reactions were electrophoresed on a nondenaturing 4% Tris-glycine polyacrylamide gel for 4 h at 150V. The gels were then dried and exposed to a PhosphorImager screen for 1–7 days before scanning on a Storm PhosphorImager. Quantitation of band intensities was performed using the ImageQuant 5.0 analysis software. Experiments using hmg2 (see Fig. 6, b and c) were also performed with hmg1 with the same trend in binding intensity between the Sox-11 mutants. All aspects of each mobility shift assay were repeated and similar results were obtained. The initial goal of this study was to elucidate the mechanisms permitting selective recruitment and activation by specific Sox proteins at a particular gene. To address this issue, we compared the function of Sox-2 with the related transcription factor Sox-11. These proteins were selected for several reasons. First, the gross domain structure of each has been studied, revealing the location of the DNA binding domain and transactivation domain of each protein (4Kamachi Y. Uchikawa M. Collignon J. Lovell-Badge R. Kondoh H. Development. 1998; 125: 2521-2532Crossref PubMed Google Scholar, 19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Second, to understand the function of a transcription factor in vivo, it is best studied in a model system using a known binding sequence from a regulated gene. The binding site for Sox-2 within the enhancer of the FGF-4 gene has been determined and provides an excellent model system (15Yuan H. Corbi N. Basilico C. Dailey L. Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (643) Google Scholar, 16Johnson L.R. Lamb K.A. Gao Q. Nowling T.K. Rizzino A. Mol. Reprod. Dev. 1998; 50: 377-386Crossref PubMed Scopus (25) Google Scholar). Thus far, target genes of Sox-11 have not been identified; however, the FGF-4 enhancer sequence has also been utilized in a partial characterization of Sox-11 (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), indicating that it may provide a relevant system in which to study this transcription factor. Finally, the expression pattern of Sox-2 and Sox-11 within the developing nervous system overlaps, leading to the postulate that there are cell types in which some genes may be regulated differentially depending on whether Sox-2 or Sox-11 binds to a cis-regulatory element (24Uwanogho D. Rex M. Cartwright E.J. Pearl G. Healy C. Scotting P.J. Sharpe P.T. Mech. Dev. 1995; 49: 23-36Crossref PubMed Scopus (349) Google Scholar). To compare Sox-2 and Sox-11 functionally, we assayed their ability to activate the pCATSO3 promoter/reporter gene construct in HeLa cells, which do not contain any known Sox-like activity (15Yuan H. Corbi N. Basilico C. Dailey L. Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (643) Google Scholar, 19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The pCATSO3 plasmid contains six tandem repeats of a 24-bp region of theFGF-4 enhancer upstream from an SV40 promoter driving the expression of a chloramphenicol acetyltransferase (CAT) reporter gene. A promoter/reporter construct containing multiple HMG binding sites was necessary for these studies because gene activation by either Sox-2 or Sox-11 via a single HMG site did not rise above the basal expression of the promoter/reporter gene construct (data not shown). The 24-bp region contains both the HMG binding site and POU binding site found in theFGF-4 enhancer. The pCATSO3 construct was employed to perform a functional comparison of Sox-2 and Sox-11 because it has been used previously to identify the transactivation domain of Sox-2 and to identify p300 as a potential coactivator of Sox-2 activity (19Nowling T. Johnson L.R. Wiebe M. Rizzino A. J. Biol. Chem. 2000; 275: 3810-3818Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Furthermore, a very similar construct, also containing the HMG/POU enhancer sequence of the FGF-4 gene, was utilized in the initial characterization of Sox-11 (20Kuhlbrodt K. Herbarth B. Sock E. Enderich J. Hermans-Borgmeyer I. Wegner M. J. Biol. Chem. 1998; 273: 16050-16057Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). For the functional comparison of Sox-2 and Sox-11, HeLa cells were transiently transfected with pCATSO3 and increasing concentrations of expression plasmid encoding either Sox-2F or Sox-11F, each tagged at their N terminus with a FLAG epitope. This revealed that Sox-11F is a much more potent activator of the pCATSO3 promoter/reporter gene construct than Sox-2F (Fig.1a). At all concentrations tested, transfection of Sox-11F led to 40–75-fold more reporter gene activity than Sox-2F. This experiment was also repeated with Sox-2 and Sox-11 expression constructs without FLAG epitopes, and no differences were observed between the ability of the tagged and untagged proteins to transactivate (data not shown). Additionally, to verify that the functional differences observed between Sox-2F and Sox-11F were not caused by variations in protein expression, Western blot analysis was performed on nuclear extracts of transfected HeLa cells using the anti-FLAG M2 antibody. This analysis revealed that there was little difference between Sox-2F and Sox-11F expression in the nucleus (Fig.1b). In fact, in multiple experiments, we found that Sox-11F expression was consistently lower than Sox-2F. Thus, the difference in transactivation capability between Sox-2F and Sox-11F (Fig.1a) may be an underestimate. Our observation that Sox-11F was such a potent activator on its own led us to question whether an endogenous POU factor could be binding at the adjacent site and assisting in Sox-11F-mediated activation. This is an important question because Sox-2 requires the partner Oct-3 for act" @default.
• W2008209085 created "2016-06-24" @default.
• W2008209085 creator A5062582917 @default.
• W2008209085 creator A5068288826 @default.
• W2008209085 creator A5088746193 @default.
• W2008209085 date "2003-05-01" @default.
• W2008209085 modified "2023-10-17" @default.
• W2008209085 title "Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity" @default.
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