interplay of sox and pou factors in regulation of the nestin

MOLECULAR AND CELLULAR BIOLOGY, Oct. 2004, p. 8834–8846 Vol. 24, No. 200270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.20.8834–8846.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Interplay of SOX and POU Factors in Regulation of the Nestin Genein Neural Primordial Cells

Shinya Tanaka,1 Yusuke Kamachi,1 Aki Tanouchi,1 Hiroshi Hamada,1 Naihe Jing,2 andHisato Kondoh1*

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan,1 and Laboratory ofMolecular Cell Biology and Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai

Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China2

Received 28 January 2004/Returned for modification 12 March 2004/Accepted 23 July 2004

Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed asmarkers for neural primordium, suggesting their regulatory link. We have identified adjacent and essentialSOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer includingthese sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimericforms in transfection assays and was utilized in assessing the specificity of the synergism; combinations ofeither group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mousespinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subven-tricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) wasexpressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderateexpression was observed in the intermediate zone, distributed in the cytoplasm. These data support the modelthat synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestinexpression. Evidence also suggests that such interactions are involved in the regulation of neural primordialcells.

Specific gene regulation for the determination of neuralprimordial cells is of high interest for its possible contributionto stem cell biology, where neural tissues are of primary im-portance. The majority of the neural primordial cells present inthe central nervous system (CNS) from embryonic to adultstages (2, 47) have been documented to express the interme-diate filament protein Nestin (11, 28, 41). Given that transcrip-tional regulation determining a cell type is largely reflected bythe regulation of genes specific to the cell type, we focused onthe regulation of the Nestin gene to better understand generegulation that is determinative for the neural primordial state.

Previous studies of rat and human Nestin genes indicatedthat Nestin expression in the CNS, at least during embryonicstages, is largely regulated by a nervous system-specific en-hancer located in the 3� half of the second intron (21, 29, 58,61). After an analysis of a 257-bp-long Nestin enhancer in therat, Josephson et al. (21) identified putative binding sites forPOU, AP2, and a hormone-responsive element; further assess-ment of their involvement in Nestin enhancer activity was per-formed using transgenic mouse embryos. Mutational alter-ations of the downstream POU factor sites, bound by class IIIPOU factors in vitro, largely eliminated the activity of theenhancer; mutations of other sites altered the domain of theCNS in which the enhancer showed activity (21). These previ-ous results indicated that the activation of the Nestin neuralenhancer is fundamentally dependent on the binding of a POU

factor to the downstream site and that other nuclear factorsmay cooperate in establishing this enhancer activity.

POU family transcription factors contain the DNA-bindingPOU domain, consisting of the POU-specific domain and ho-meodomain, and are grouped into six classes (4, 19, 27). Theclass III POU factors, Brn1, Brn2, Brn4, and Oct6 (also calledTst1/SCIP), are widely expressed in the developing CNS, withextensive regional overlap (1, 18), and presumably share re-dundant functions. Corroborating this notion, single-knockoutmice lacking Brn1, Brn2, Brn4, or Oct6 do not have significantneural phenotypes, and only the simultaneous loss of Brn1 andBrn2 in double-knockout mice causes a mild CNS phenotype(4, 30, 46).

Transcription factors expressed in tight association with theneural primordia are group B1 SOX proteins, represented bySOX2 and including SOX1 and SOX3 (8, 37, 40, 45, 51, 57).SOX proteins have similar DNA-binding HMG domains (24,36, 53) and are classified into groups A through J according tothe characteristics of the HMG domain and extra-HMG do-main sequences (6).

Early neurogenesis in a variety of vertebrate systems is as-sociated with expression of group B1 Sox genes (31, 37, 45, 49).In the chicken, an enhancer for Sox2 that directly responds toneural induction signals has been identified and has beenshown to be conserved among amniotes (49). In Xenopus,forced expression of Sox2, in combination with basic fibroblastgrowth factor, initiates the neural development of the ecto-derm (31). It has also been demonstrated that group B1 SOXactivity is involved in maintaining the neural primordia in theembryonic neural tube (7, 15). Taken together, these observa-tions indicate that the expression of group B1 SOX proteins is

* Corresponding author. Mailing address: Graduate School of Fron-tier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7963. Fax: 81-6-6877-1738. E-mail:[email protected].

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directly involved in establishing and maintaining neural pri-mordial cell populations.

SOX and POU transcription factors often regulate genesinterdependently, forming a complex on the DNA regulatorysite (12, 24). In this study, we demonstrate that group B1 andgroup C SOX proteins interact with POU factors and activatethe Nestin neural enhancer. In the embryonic spinal cord, Nes-tin expression occurs in the cells expressing group B1 SOX andBrn2 in their nuclei (ventricular zone) and in those expressinggroup C SOX and Brn2 in the nucleus (subventricular zone).In the intermediate zone, the cells coexpress SOX11 and Brn2;however, Brn2 is largely sequestered in the cytoplasm. Thus,the participation of the POU factors in transcriptional regula-tion is determined by their subcellular localization. This regu-lated interaction between transcription factors presumably un-derlies the mechanisms that the determine the neuralprimordial cell state.

MATERIALS AND METHODS

Immunohistology. Mouse embryos were fixed with 4% paraformaldehyde–phosphate-buffered saline and cryosectioned at a thickness of 10 to 15 �m. Aftertreatment with 10% serum of the animal species used for secondary antibodies,sections were incubated with primary antibodies and then with Alexa Fluor488-conjugated anti-rabbit immunoglobulin G and/or Alexa Fluor 568-conju-gated anti-mouse immunoglobulin G (Molecular Probes). Observations weremade using a Zeiss LSM5 Pascal laser microscope. Anti-SOX2 antibodies (dem-onstrating no cross-reactivity with SOX1 or SOX3) were produced in a rabbit byinjecting the SOX2 N-terminal 19-amino-acid peptide linked to keyhole limpethemocyanine; antibodies were affinity purified using peptide-conjugated beads.Anti-group B1 SOX antibodies (with demonstrated preference for SOX1 andSOX3 over SOX2 and indicated as anti-SOX1/[2]/3 in the figures) were derivedfrom a rabbit similarly immunized with the SOX3 C-terminal 15 peptides. Anti-SOX11 and anti-Brn2 antibodies were obtained from Santa Cruz Biotechnology.Mouse anti-Nestin monoclonal antibody was obtained from BD Pharmingen.

Embryo electroporation. Enhancer sequences were inserted into the XhoI siteof the ptkEGFP vector plasmid, delivered to the area of the caudal neural foldof a stage 10 chicken embryo, and electroporated as previously described (49).

cDNAs, cell culture, and transfection. Full-length Nes258 enhancer and oc-tamerized Nes30 sequences were inserted into p�51LucII (22) reporter vectors.The SOX and POU proteins were expressed by inserting cDNAs into the vectorpCMV/SV in transfected cells (22). Both mouse and chicken cDNAs for SOX1,-2, -3, and -11 were used (DDBJ accession numbers for mouse sequences,AB108672, AB108673, AB108674, and AB108675), yielding essentially identicalresults. However, since data for SOX9, SOX14, and SOX21 were obtained byusing chicken cDNAs, the data shown in the figures were generated usingchicken SOX cDNAs (22, 23) for consistency. POU factor cDNAs were of mouseorigin (16, 34, 35).

Liver cells from a 10-day-old chicken embryo were transfected by a calciumprecipitation method (22) with 1.5 �g of DNA containing p�51LucII reporter(1.3 �g), pCMV/SV effecter (0.1 �g), and reference pMiwZ (52) (0.1 �g). Braincells from a 7-day-old chicken embryo and other cells, including COS7, 10T1/2,and MNS70 (32), were transfected with 3 �l of Fugene6 (Roche) and 1 �g ofDNA containing 0.9 �g of reporter and 0.1 �g of reference vectors. Transfectionwas done at least in triplicate, and the average luciferase expression is indicated(with standard errors) in the figures.

Analysis of transcripts using reverse transcription (RT)-PCR. Total RNAswere prepared from MNS70 or mouse embryonic spinal cords using a RiboPurekit (Ambion). cDNAs were synthesized from total RNA using ThermoScriptreverse transcriptase (Invitrogen) and oligo(dT) primer and amplified by PCRusing ExTaq polymerase (TaKaRa) and the following sets of forward (F) andreverse (R) primers: Nestin, F (CGCTGGAACAGAGATTGGAAGG) and R(GTCTCAAGGGTATTAGGCAAG); Sox2, F (ACCTACAGCATGTCCTACTCG) and R (GGGCAGTGTGCCGTTAATGG); Sox11, F (TCAGCTGCTGAGGCGCTACAG) and R (GAACACCAGGTCGGAGAAGTTCG); Brn2, F(GGCGCCGAGGATGTGTATGG) and R (CATTCTACTTCATTGCCTGGG); Brn4, F (ACAGCTGCCTCGAATCCCTAC) and R (ATGCAGGCTCTGTGTGGAGG); and �-actin (32), F (TGCCCATCTATGAGGGTTACG) and

R (TAGAAGCATTTGCGGTGCACG). The PCR products were electropho-resed and stained with SYBR GREEN I (Molecular Probes).

EMSA and protein coprecipitation. Recombinant proteins, tagged with gluta-thione S-transferase (GST) or maltose binding protein (MBP) at the N terminusand oligohistidine at the C terminus, were synthesized in Escherichia coli andaffinity purified according to the method of Kamachi et al. (25). For electro-phoretic mobility shift assays (EMSA), the N-terminal GST tag was removedafter purification. Conditions for EMSA and protein coprecipitation assays usingimmunoblotting were previously described by Kamachi et al. (25). Dissociationconstants (Kd) for the complexes formed between the recombinant protein andthe probe DNA were estimated based on the following relationships: Kd �(�[total protein input] � [bound protein]) [free probe]/[bound probe], where �indicates the active fraction of the recombinant protein. The concentrations oftotal protein input and bound protein, as well as those of free and bound probes,were directly determined from experimental data. By total protein input varia-tions, Kd and � were estimated. The Kd for SOX2(3-202) bound to Nes30sequence was estimated to be 1.0 10�8 M (� � 0.75); the Kd for MBP-Brn2(215-445) bound to Nes30 was 3.6 10�10 M (� � 0.58).

RESULTS

Mouse Nestin gene has a 258-bp neural enhancer dependenton binding sequences for SOX and POU factors. It has beendemonstrated in the rat gene that Nestin expression in theneural tube is determined by an enhancer, 257 bp long, locatedin the second intron; the activity of the enhancer is dependenton the integrity of a small segment, including a POU factorbinding site (21). We have found a 258-bp sequence in themouse Nestin second intron that is almost identical (96% iden-tity) to the rat neural enhancer (Fig. 1A). It is known that thesecond intron in the human Nestin gene also contains a con-served CNS-active enhancer (29), which is 75% identical to themouse sequence in the corresponding 256-bp region. The es-sential binding site for a POU factor in the enhancer is con-served among these animal species (Fig. 1B).

Considering the association of group B1 SOX and Nestinexpression in the neural primordial cells, we searched for apotential SOX binding site in the 258-bp mouse Nestin en-hancer (Fig. 1B). A candidate SOX binding sequence, GACAAAA, was found immediately upstream of the POU bindingsite and is perfectly conserved in the rat Nestin enhancer. In thehuman Nestin enhancer sequence, GACAAAA is altered toGACAATG; however, the latter sequence also provides agood binding site for group B1 SOX (24).

To confirm that the 258-bp sequence of the mouse Nestingene has nervous system-specific enhancer activity, and to eval-uate the potential contributions of SOX and POU factors tothis activity, we took advantage of electroporation of chickenembryos. Wild-type or mutated 258-bp sequences were placedupstream of the herpes simplex virus thymidine kinase (tk)promoter of the tk-EGFP gene (49), and chicken embryos atstage 10 were electroporated at the caudal neural plate withthis gene and an HcRed1 expression vector. The embryos wereobserved after 48 h at stage 21 (Fig. 1C). When the wild-typesequence was employed, the EGFP reporter gene was stronglyexpressed in the spinal cord (Fig. 1C, a), while coelectropo-rated HcRed1 was expressed in the spinal ganglia and epider-mis as well (Fig. 1C). When either of the mutations, M1 or M2(disrupting the binding sites for SOX and POU, respectively),was introduced into the 258-bp sequence, enhancer activity wasattenuated (Fig. 1C, c and e). These observations indicate thatthe 258-bp sequence has nervous system-specific enhancer ac-

VOL. 24, 2004 SOX-POU SYNERGISM IN NEURAL PRIMORDIA 8835

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8836 TANAKA ET AL. MOL. CELL. BIOL.

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tivity and that the integrity of both binding sites is essential forthe enhancer.

To further confirm the dependence of the Nestin enhanceron SOX and POU binding sites, we tested the activity of theenhancer in the neural stem cell line MNS70 (32). Placing thewild-type Nestin enhancer upstream of a reporter luciferasegene (Fig. a) activated luciferase expression in transfectedMNS70 cells 75-fold (Fig. 1D, b), indicating that endogenoustranscription factors are sufficient for activation of this en-hancer. Mutations M1 and M2, disrupting the binding sites forSOX and POU factors, respectively, significantly reduced theactivity to a 30- to 40-fold activation level, thus confirminginvolvement of these factors in generating the activity. Exoge-nous expression of SOX2 or Brn2 augmented the activationlevel by the wild-type enhancer to 85-fold, and their simulta-neous expression increased it further to 100-fold activation,indicating that these SOX and POU factors synergistically ac-tivate the Nes258 enhancer and that MNS70 cells containnearly saturating levels of these or analogous factors. Indeed,MNS70 cells expressed Sox2 (group B1), Sox11 (group C), andBrn2/4 (class III) at a level even higher (except for Brn4) thanmouse spinal cords of embryonic day 10.5 (E10.5) and E13.5embryos, as assessed by RT-PCR (Fig. 1D, c). However, usingmutant enhancers carrying either mutation M1 or M2, exoge-nous supply of SOX2 and/or Brn2 had no effect on the expres-sion of luciferase reporter (Fig. 1D, b), indicating that thesetranscription factors act only in combination in activation ofthe Nes258 enhancer.

The Nestin core enhancer, composed of SOX and POU bind-ing sites, can be activated by the synergistic action of SOX2and Brn2. Many cell-specific enhancers are composed of bind-ing sites for cell-specific nuclear factors and those for factorswith broader specificity (reference 14 and references therein).The core regulatory region of the enhancers provides bindingsites for specific factors (25), generally essential for the entireenhancer activity, and the multimeric form demonstrates itsintrinsic enhancer activity (reference 14 and referencestherein).

In the case of the Nestin enhancer, the SOX-POU bindingregion may have the property of the Nestin enhancer core

sequence; the potential enhancer activity may be demonstratedby its multimerization. Thus, the 30-bp region (Nes30) of theNestin enhancer containing SOX and POU factor binding sites(Fig. 1B and 2A) was octamerized and placed upstream of the�51LucII reporter gene (22) (Fig. 2A). The constructs werethen transfected into different cultured cells to determinewhether SOX-POU-dependent enhancer activity is detectable(Fig. 2B and C).

The octamerized Nes30 sequence [Nes30(8-mer)] signifi-cantly activated luciferase expression in primary brain cells;however, this activation was absent in liver cells (Fig. 2B andC). Among the cell lines tested, the neural stem cell lineMNS70 (32) supported enhancer activity of the Nes30(8-mer)sequence. In nonneural COS7 or 10T1/2 cells, the Nes30 se-quence showed no activity (Fig. 2B and C). The activation ofluciferase expression by Nes30(8-mer) in the brain cells and inthe neural stem cells was compromised when the sequence hadeither the M1 or M2 mutation (Fig. 2B). These results indicatethat the Nes30 sequence has nervous system-specific core en-hancer activity and that this activity presumably depends onsimultaneous binding of SOX and POU factors.

To establish the contribution of SOX and POU transcriptionfactors in the regulation of the Nes30 core enhancer, we ex-ogenously expressed SOX2 and Brn2 (class III POU) in livercells, where the core enhancer has no activity (Fig. 2). WhenSOX2 was expressed alone, the cotransfected Nes30(8mer)-carrying luciferase gene was not activated, whereas singularBrn2 expression caused only very weak activation (5- to 10-fold). By contrast, when SOX2 and Brn2 were coexpressed,there was dramatic activation of the cotransfected luciferasegene (90- to 100-fold) (Fig. 3B). This strong activation broughtabout by the coexpression of SOX2 and Brn2 was extinguishedwhen mutations M1 (SOX2 binding site) or M2 (POU bindingsite) were introduced into the Nes30 sequence (Fig. 3C). Thus,the Nes30 enhancer is activated by the synergistic action ofSOX2 and Brn2 bound to the sequence. (Low activation ofluciferase expression by exogenous Brn2 alone, in spite of itstotal dependence on the integrity of the SOX binding site, isnow ascribed to low-level endogenous expression of SOX11 in

FIG. 1. Involvement of SOX and POU binding sites in activation of the Nestin neural enhancer. (A) Potential SOX2 binding site found adjacentto the POU factor binding site of the Nestin neural enhancer. The 258-bp-long enhancer sequence in the second intron of the mouse Nestin gene,which is similar to rat and human enhancers, is schematically shown. The blue and red boxes represent the potential SOX binding site and the POUfactor binding site, respectively. (B) Nucleotide sequence of a portion of the mouse Nestin enhancer in the gray box in panel A, which is alignedwith the corresponding sequences of rat and human. Gray shading indicates conserved nucleotide sequences. The M1 mutation disrupts SOXbinding motifs (24, 36, 53) and abolishes SOX2 binding (Fig. 4), while mutation M2 has been shown to eliminate Nestin neural enhancer activity(21). The mutated sequences are shaded in purple. The Nes30 core sequence used in the following analyses is also indicated. (C) Activity of themouse Nestin neural enhancer (Nes258) and its M1-M2 mutant forms in the electroporated spinal cord of a chicken embryo. Expression vectorsfor enhancer-dependent EGFP (Reporter) and HcRed1 (Reference) are indicated on top. Electroporation was performed at stage 10, andobservations were made after 48 h at stage 21. The HcRed1 reference vector was also included in electroporation. (a, c, and e) EGFP fluorescencein the spinal cord representing activity of Nestin enhancer. (a) Wild-type (WT) enhancer was strongly active in the spinal cord. (c and e) Themutation-containing enhancer, either M1 (c) or M2 (e), was totally inactive. (b, d, and f) HcRed1 fluorescence superimposed on bright-fieldimages. (D) Activity of Nes258 enhancer and its dependence on the SOX and POU binding sites in the neural stem cell line MNS70 and effectof exogenous SOX2 and/or Brn2 expression on the activity. (a) Structure of Nes258 enhancer-bearing luciferase vector and expression vector forSOX2 or Brn2 effectors. CMV, cytomegalovirus. (b) Nes258 enhancer-bearing luciferase vector was transfected into MNS70 neural stem cells,displaying 75-fold activation compared to the enhancer-free vector. Exogenous expression of SOX2 or Brn2 singly augmented the level ofactivation to roughly 85-fold, and that of SOX2 and Brn2 together activated luciferase expression nearly 100-fold. Using mutant enhancers withdefective SOX (M1) or POU (M2) binding sites, activation of the luciferase reporter fell in the range of 30- to 40-fold, and this was not augmentedby exogenous SOX2 and/or Brn2 any further. The luciferase activity level generated by the reporter in the absence of enhancer was taken as 1.(b) Expression of Nestin, Sox, and Pou factor genes in the MNS70 line demonstrated by RT-PCR, in comparison with the embryonic spinal cord.


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liver cells [data not shown], which was later found to synergizewith Brn2 in activation of the Nes30 enhancer [see below].)

Binding of SOX2 and Brn2 to the Nes30 sequence in vitro.To confirm that SOX2 and Brn2 bind to the Nes30 sequencetogether, electrophoretic mobility shift assays (EMSA) wereperformed using the Nes30 sequence probe.

The region from amino acids 3 to 202 of SOX2, containingthe HMG domain, and the region from amino acids 215 to 445of Brn2, containing the POU domain, were both tagged withthe MBP, synthesized (Fig. 4A), and used in EMSA (Fig. 4B).The individual protein domains bound efficiently to the probe(Fig. 4B, lanes 1, 2, 4, and 5), and the combination of theseproteins yielded a new, slowly migrating band, indicating si-multaneous binding of the two proteins (Fig. 4B, lane 3). Withthe increase of MBP-Brn2(215-445), the slowly migrating bandwas intensified in a predictable manner from noncooperative,stoichiometric binding of these proteins to the probe (Fig. 4B,

lane 6). A dissociation constant of 1.0 10�8 M was estimatedfor binding of SOX2(3-202) to the Nes30 probe, and 3.6 10�10 M for binding of Brn2(215-445) to the probe, as de-scribed in Materials and Methods (data not shown). MutationM1 eliminated binding of SOX2(3-202), while mutation M2destroyed binding of MBP-Brn2(215-445) (Fig. 4B, lanes 7 to12).

Given that SOX2 and Brn2 act synergistically in the activa-tion of the Nes30 core enhancer, it is possible that these pro-teins interact directly without the intervention of DNA. Suchdirect interaction between SOX2 and Oct3/4, as well as that ofSOX2 and Pax6, has been demonstrated (3, 25). To test this,GST-tagged SOX2(3-202) protein was attached to glutathione-Sepharose beads and examined to determine if MBP-taggedBrn2(215-445) coprecipitated (Fig. 4C). Attempts at synthesiz-ing full-length proteins were hampered by extensive proteolysisduring sample preparation, but previous reports indicated pro-tein interaction sites close to HMG and POU domains (3).GST-SOX2(3-202) coprecipitated with MBP-Brn2(215-445)with an efficiency comparable to that of MBP-Pax6(1-169),which is known to bind SOX2(3-202) (25). No such bindingwas detected when GST alone, or MBP linked to an unrelatedprotein (MBP-LacZ�), was employed. The observations indi-cate that SOX2 and Brn2 proteins interact without Nes30DNA. More extensive protein-protein interactions might takeplace if full-length proteins are involved. Thus, protein-proteininteraction may participate in the synergistic action of SOX2and Brn2 in activating the enhancer.

Comparison with other cases of SOX2-POU interaction onregulatory DNA sequences, mainly involving Oct3/4, indicatesthat the arrangement of binding sites of SOX2 and Brn2 in theNes30 sequence is unique, and individual binding sites areinverted relative to other cases (3, 13, 33, 48, 59) (Fig. 5A). Inaddition, the distance between the SOX and POU binding sitesis larger in the Nestin enhancer (6 bp between the binding sitescompared to 3 [Fgf4] or 0 [all other cases] bp). To see whetherthis arrangement of binding sites is essential for Nes30 en-hancer activity, the binding site sequences were swapped(Nes30-Swap), or the space between the binding sites wasincreased by the insertion of 10 bp (Nes30-Ins). The alterationsof the sequence did not affect binding of SOX2 and Brn2 (Fig.5B). With these rearrangements, however, the Nes30 sequencelost the enhancer activity in SOX2/Brn2-transfected liver cells(Fig. 5C). The Nes30-Swap sequence is analogous to one of theFgf4 enhancer mutants lacking the activity due to increasedspacing between the SOX2 and POU binding sites (3). Thus,the particular orientation and spacing between the SOX2 andPOU binding sites found in the Nes30 sequence represents anew functional arrangement of binding sites activated by SOX2and a POU factor.

Differential regulation of the Nestin enhancer by variouscombinations of SOX and POU factors. In addition to SOX2and Brn2, various SOX and POU factors are expressed in theembryonic CNS, as assessed by in situ hybridization of the genetranscripts. All group B1 Sox genes are expressed in the ven-tricular zone, while Sox2 is additionally expressed in the roofand floor plates (37, 40, 50, 51, 57). Of the group B2 Sox genesSox14 and Sox21, the latter is expressed primarily in the ven-tricular zone (50). Additionally, the group C Sox genes Sox11,Sox4, and Sox12 are strongly expressed in the subventricular

FIG. 2. Activities of the 30-bp core enhancer (Nes30) in severalcultured cells. (A) Structures of the reporter plasmid (left) and wild-type (WT), M1 (SOX2 site mutant), and M2 (POU site mutant) Nes30sequences. Altered nucleotides are shaded. Lowercase letters indicatelinker sequences. An octamerized Nes30 sequence was inserted intothe p�51LucII reporter plasmid (22) in both normal (N) and reversed(R) orientations relative to the direction of transcription. (B) Activitiesof Nes30-WT, M1, and M2 in chicken primary brain cells and MNS70neural stem cells. C. Activities of Nes30-WT in chicken primary livercells, COS7 (kidney) cells, and 10T1/2 fibroblasts. The luciferase ac-tivity level generated by the reporter in the absence of enhancer wastaken as 1.


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and intermediate zones (9, 17, 20, 26, 51). The SOX9 (groupE) protein has been shown to be expressed in the nuclei of theventricular zone and glial precursors (44).

Among POU family transcription factor genes, those codingfor class III factors, Brn1/Brn2/Brn4/Oct6, are expressed in theventricular through intermediate zones in overlapping butslightly different anteroposterior domains of the CNS (1, 4, 18).The class V POU Oct3 (or Oct4) is expressed throughout theneural plate in very early embryos (38, 42). The class II POUprotein gene Oct2 is expressed in a regionally restricted fashion(27), while the class VI POU gene Brn5 (Emb/Cns-1) is ex-pressed throughout the CNS (10).

To learn how these SOX and POU factors may cooperate inregulating Nestin expression, analogous to SOX2 and Brn2,various combinations of SOX and POU factors were expressedin liver cells and their effects on the activity of the Nes30(8-mer) enhancer was assessed (Fig. 6).

In combination with Brn2, all group B1 SOX proteins,SOX1, SOX2, and SOX3, activated the Nes30(8-mer) en-hancer nearly 100-fold, confirming analogous activities of thesefactors. The group B2 SOX proteins, SOX14 and SOX21, orgroup E SOX9, did not activate the Nes30 enhancer (Fig. 6A).

Importantly, group C SOX11, in combination with Brn2,

also activated the Nes30(8-mer) to a significant extent (30- to40-fold activation) (Fig. 6A). This contrasts with activation ofthe �-crystallin DC5 enhancer by cooperation of SOX2 withPax6, where SOX11 could not replace SOX2 (22, 25).

Among various POU factors which were examined for thepotential to activate the Nes30(8-mer) enhancer in combina-tion with SOX2 (Fig. 6B), class III POU proteins (Brn1, Brn2,Brn4, and Oct6) strongly activated the enhancer, in whichactivation by Brn1 was relatively moderate. Oct3/4 (class V), incombination with SOX2, activated the enhancer comparably toclass III POU factors. Class II Oct2 protein caused weak ac-tivation, whereas class VI Brn5 showed no activation withSOX2.

Expression of group B1 SOX, SOX11, and Brn2 in thespinal cord and its relationship with Nestin expression. Theobservation described above indicates that group B1 and groupC SOX proteins (at least SOX11) cooperate with class III POUfactors and activate the Nestin enhancer through binding to theNes30 sequence. To correlate the synergism of SOX and POUfactors that occurs on the Nes30 sequence with the expressionof Nestin in vivo, we examined the expression of group B1SOX, SOX11, and Brn2 proteins in the embryonic spinal cordby immunostaining histological sections.

FIG. 3. Synergistic activation of the Nes30 enhancer by exogenous SOX2 and Brn2. (A) Schematic structure of the reporter and SOX2/Brn2-expressing effecter plasmids. The effecter plasmids had full-length cDNAs of SOX or POU factors, which are transcribed from the cytomegalovirus(CMV) promoter. (B) Activation of the Nes30 enhancer by SOX2 and Brn2. Various amounts of effecter plasmids designed to express SOX2 orBrn2, and the reporter plasmids, were transfected into liver cells. (N) and (R) indicate the orientation of the Nes30 enhancer in the reporterplasmid (normal and reversed, respectively). (C) Effect of mutations in the SOX binding site or POU factor binding site on Nes30 enhancer activity.The enhancer was in N orientation. The error bars indicate standard errors.


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At E10.5, when virtually all cells in the neural tube expressedNestin (Fig. 7Aa) and were largely mitotic (37), SOX2 wasexpressed in the major population of cells and localized to thenuclei (Fig. 7Ab and Ba), except for the most external regioncorresponding to the forming subventricular zone, whereSOX11 was strongly expressed (Fig. 7Ad and Bb). At this stageof spinal cord development, expression of SOX2 and SOX11overlap in a large fraction of cells (Fig. 7B and C). Immuno-staining with antibodies detecting all group B1 SOX proteins(with some preference to SOX1 and SOX3) showed essentiallythe same distribution of the antigen, indicating that expressiondomains of other group B1 SOX proteins are included in thatof SOX2 (Fig. 7Ac and Bd). On the other hand, Brn2 wasdistributed in all nuclei (Fig. 7Ae and Be), surrounded byNestin-expressing cytoplasm (Fig. 7Bf).

In E13.5 spinal cord, SOX2 expression was confined to theventricular zone (Fig. 8Ab and Ba), as with other group B1SOX proteins (Fig. 8Ac and Bc). SOX11 expression at thisstage was strong in the subventricular zone, extending into theintermediate zone with a moderate expression level (Fig. 8Adand Bb), thus making a boundary with the SOX2-expressingventricular zone (Fig. 8Bd).

Immunostaining with anti-Brn2 antibodies indicated wide-spread Brn2 protein expression in the neural tube. Expressionwas strong in the ventricular and subventricular zones andmoderate in the intermediate zone (Fig. 8Ae). Examination athigher magnification revealed a remarkable transition in thesubcellular localization of this protein. In the ventricular andsubventricular zones, Brn2 protein was located mostly in thenuclei, whereas in the intermediate zone, its majority localiza-tion was to the cytoplasm (Fig. 8Be and C). Thus, functionalcooperation of group B1 SOX and Brn2, and that of SOX11and Brn2, can take place in the ventricular and subventricularzones, respectively, based on their nuclear colocalization. Inthe intermediate zone, the cooperation is likely inhibited be-cause of the cytoplasmic localization of Brn2. This accounts forexpression of Nestin in the cells of the ventricular zone (groupB1 SOX and nuclear Brn2), and those of the subventricularzone (SOX11 and nuclear Brn2).

DISCUSSION

SOX- and POU-dependent Nestin enhancer and its coresequence. To better understand gene regulation taking place inneural primordial cells, we focused on the regulation of theNestin gene marking these cells (11, 38, 41, 43). Our study wasbased on the idea that transcriptional regulation determining acell type is largely reflected by the regulation of genes specificto that cell type.

In addition to the Nestin gene, expression of group B1 Soxgenes, represented by Sox2, is also regarded as an indication ofthe neural primordial cells (8, 43, 49, 60). Indeed, recent stud-ies that indicated interference with group B1 SOX function inthe embryonic spinal cord demonstrate the function of thisclass of SOX proteins in maintaining the neural primordialstate (7, 15). We speculated that there would be a direct reg-ulatory link between expression of Nestin and group B1 Soxgenes and thus investigated the Nestin enhancer.

Previous studies identified the nervous system-specific en-hancer of the Nestin gene in the second intron (29, 61), the

FIG. 4. Binding of SOX2 and Brn2 to Nes30 DNA and their pro-tein-protein interaction. (A) Recombinant SOX2(3-202) andBrn2(215-445) were synthesized as fusion proteins with GST andMBP, respectively. (B) Binding of SOX2(3-202) and MBP-Brn2(215-445) with GST removed to wild-type (WT) or mutant (M1 and M2)forms (Fig. 1B) of the Nes30 sequence in vitro, as assessed by EMSA.The recombinant proteins bind to the Nes30-WT sequence efficiently,either alone (lanes 1 and 2) or in combination (lane 3). In lane 3, aslowly migrating band representing a SOX2(3-202)/MBP-Brn2(215-445)/probe ternary complex is observed. A larger amount of Brn2(215-445) increased band intensity, and the formation of the ternary com-plex was stoichiometric (lanes 4 to 6). Mutation M1 attenuated bindingof SOX2(3-202) (lanes 7 and 9), while mutation M2 disrupted thebinding of Brn2(215-445) (lanes 11 and 12). (C) Protein interaction ofSOX2 and Brn2 without Nes30 DNA. MBP-tagged proteins, LacZ�,Brn2(215-445), and Pax6(1-169), were incubated with GST-SOX2(3-202) or GST and coprecipitated with glutathione-Sepharose, and theprecipitated MBP fusion proteins were detected by immunoblottingusing anti-MBP antibodies.


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activity of which is totally dependent on the binding of class IIIPOU factors exemplified by Brn2 (21). It has also been indi-cated that other transcription factors, e.g., those binding tohormone-responsive elements, have subsidiary roles in aug-menting activity of the enhancer in specific domains of theCNS (21, 29, 58). In this study, we demonstrated that SOXproteins binding to a site immediately upstream of the POUsite are also essential for activation of the Nestin enhancer (Fig.1 to 3), providing the functional link between the group B1SOX and Nestin expression.

Recombinant SOX2 and Brn2 proteins bind to these sites(Fig. 4), and mutational abolition of the SOX binding site (M1)eliminated the activity of the full-length (258-bp) Nestin en-hancer in electroporated chicken spinal cord and in the trans-fected neural stem cell line MNS70. The effect of the SOX sitemutation was similar to that of POU site mutation (M2), sup-porting the synergistic action of SOX and POU for activationof the Nestin enhancer (Fig. 1C and D).

Many cell-type- or tissue-specific enhancers are comprisedof elements with narrow specificity with essential functions(and hence called “core”) and those with broader specificity.Although individual elements do not necessarily show signifi-cant enhancer activity, their regulatory potential and specificitycan be demonstrated when they are multimerized (reference14 and references therein). The 30-bp-long region of the Nestinenhancer (Nes30), including the SOX and POU binding sites,had the features of such a core element. Indeed the octamer-ized Nes30 sequence showed neural specificity and absolutedependence on the SOX and POU binding sites, providing ahandle on the synergistic interaction of the SOX-POU proteins(Fig. 2).

Synergistic function of SOX and POU proteins in regulationof the Nestin core enhancer. Taking advantage of the octamer-ized Nes30 core sequence, we demonstrated that specific com-binations of SOX and POU proteins, group B1 SOX and classIII POU and group C SOX and class III POU, are involved inthe activation of the Nestin enhancer. Although only SOX11among group C SOX factors was studied here, other group CSOX factors, i.e., SOX4 and SOX12 (also called SOX22),which share many protein motifs and are known to be ex-pressed in the subventricular and intermediate zones of theembryonic spinal cord (9, 20, 51), likely have analogous poten-tials for synergism. The specificity of the synergistic interactionis confirmed by the total absence of transactivation when

FIG. 5. Effects of the arrangement of SOX and POU factor bindingsites on the enhancer activity of Nes30. (A) Nucleotide sequences andarrangements of SOX2 and Oct3/4 binding sites found in the enhanc-ers of Fgf4 (3), UTF1 (33), Sox2 (48), and HoxB1 (13) and comparisonof those with SOX2 and POU binding sites in the Nes30 sequence.SOX2 and POU binding sequences are indicated by blue and redarrows, respectively. The orientations of both SOX2 and POU sites inthe Nes30 sequence are in reverse, compared with the four enhancersindicated above. Two mutant sequences of Nes30 for the arrangementof SOX2 and POU binding sites were prepared. The Nes30-Swapmutant enhancer had the binding sequences in inverted orientations,where the inverted SOX binding sequence is underlined. The Nes30-Ins mutant enhancer had 10 bp inserted between SOX and POU factor

binding sites. (B) Binding of SOX2(3-202) and Brn2(215-445) to theSwap and Ins mutant sequences of Nes30. SOX2(3-202) (lanes 1, 4,and 7), MBP-Brn2(215-445) (lanes 2, 5, and 8), and the combination ofSOX2(3-202) and MBP-Brn2(215-445) (lanes 3, 6, and 9) were mixedwith the wild-type (WT) (lanes 1 to 3), Swap mutant (lanes 4 to 6), andIns mutant (lanes 7 to 9). Nes30 probes and their binding were ana-lyzed by EMSA. (C) Effects of Swap and Ins mutations on enhanceractivity of Nes30 in transfected liver cells. Various amounts of plasmidsto express SOX2 or Brn2 were transfected to liver cells, together witha luciferase-encoding reporter plasmid carrying an octamerized en-hancer sequence. (N) and (R) indicate the orientation of the Nes30enhancer in the reporter plasmid (normal and reversed, respectively).The Swap and Ins mutations both inactivated enhancer activity. Theerror bars indicate standard errors.


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SOX14/21 (group B2), SOX9 (group E), or Brn5 (class VI) wasinvolved.

It is common for SOX proteins to cooperate with otherfactors by binding to a nearby site of target DNA sequences fortheir action and to employ distinct partner factors in differentcells, and thus to selectively regulate a cell-specific group ofgenes (12, 24, 25, 56). For instance, several enhancers active inembryonic stem (ES) cells are activated by the combinatoryaction of SOX2-Oct3/4 (3, 13, 33, 48, 59) (Fig. 5A). Previousstudies employing the Fgf4 enhancer as a model system showedthat the natural pair SOX2 and Oct3/4 expressed in ES cells(3), as well as an artificial SOX11-Brn2 pair, activated theenhancer (26); the SOX2-Brn2 pair, however, failed to do so(54), supporting the model of differential and binding site-dependent SOX-POU interactions.

The arrangement of the SOX and POU factor binding sitesequences in the Nes30 region was unique among known SOX-POU synergistic sites, including those of the Fgf4 enhancer

(Fig. 5A). Insertion of a 10-bp sequence between the SOX andPOU binding sites of Nes30, or the swapping of these sites inthe 30-bp stretch, completely inactivated the core enhancer(Fig. 5C), although individual proteins bound to these se-quences (Fig. 5B). Selective combinations of SOX and POUproteins in transactivation through binding to their uniqueconfiguration of the Nes30 sequence extend and confirm thediverse regulatory functions generated by the combined actionof SOX and POU proteins binding to adjacent sites.

It is conceivable that there are various interactions betweenSOX and POU proteins to elicit gene activation synergistically,presumably employing different interfaces of the SOX andPOU proteins. In support of this model, recent crystallo-graphic analysis of SOX2-POU complexes indeed indicatesrecruitment of distinct interaction sites of SOX2 and Oct3/4,depending on the binding site sequences (39, 55).

Regulation of SOX and POU factors in embryonic spinalcord. As the SOX and POU binding sites included in the Nes30

FIG. 6. Selective combination of SOX and POU factors in activation of the Nes30 enhancer. Various amounts of plasmids to express SOX orPOU factors were transfected into liver cells, together with a luciferase-encoding reporter plasmid carrying the octamerized enhancer sequence.(N) indicates the normal orientation of the Nes30 enhancer in the reporter plasmid. (A) Combinations of Brn2 with different SOX proteins. SOX1,SOX2, and SOX3 (group B1) strongly activated the enhancer, and SOX11 (group C) moderately activated the enhancer, while SOX9 (group E)and SOX14 and -21 (group B2) showed no activation. WT, wild type. The error bars indicate standard errors. (B) Combination of SOX2 withdifferent POU factors. All class III POU factors (although Brn1 was relatively inactive) and class V Oct3/4, when combined with SOX2, activatedthe Nes30 enhancer by nearly 100-fold, whereas class II Oct2 did so moderately. In contrast, Brn5 did not activate the enhancer.


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sequence are essential for the entire activity of the Nestinneural enhancer (Fig. 1B and C), the functional synergism ofSOX and POU factors that takes place on the Nes30 sequencemust, to a large degree, reflect the major transcriptional reg-ulation of the Nestin neural enhancer. To validate this, theexpression of transcription factors in the embryonic spinal cordwas investigated.

Previous studies using in situ hybridization indicated tran-script localization for these transcription factors: expression ofSox1/2/3 in the ventricular zone (40, 50, 51) and high expres-sion of Sox11 in the subventricular zone (9, 51). Other group CSox genes, i.e., Sox4 and Sox12, are also expressed in the CNSdomains, including the subventricular zone (9, 20, 26).

It has also been shown, by using an in situ hybridizationtechnique, that class III POU factor genes are widely expressedin the ventricular and subventricular zones of the embryonicCNS. Their spatial expression covers slightly different CNSareas along the A-P axis, but extensive overlap exists betweenthem, presumably exerting redundant functions (1, 4, 18). Thefunctional redundancy of the class III POU factors in the CNSis supported by the lack of major neural phenotypes in thesingle and double mutants lacking Brn1 and Brn2 (4, 30, 46).

Immunohistological detection of group B1 SOX and Brn2proteins carried out in this study (Fig. 7 and 8) clearly indicatedtheir spatial relationships. At E10.5, the majority of the cellshad characteristics of the ventricular zone and expressedSOX1/2/3, and an emerging subventricular zone expressing

SOX11 was not well separated (Fig. 7). Brn2 was expressedand distributed throughout the nuclei of the spinal cord (Fig.7). Nestin was also expressed in all cells, consistent with themodel of synergic interaction between group B1 SOX and classIII POU, and that of group C SOX and class III POU, inactivating Nestin expression.

At E13.5, Nestin expression was confined to those cells hav-ing their nuclei in the ventricular and subventricular zones(Fig. 8). At this stage, ventricular and subventricular zoneswere distinct as a zone of group B1 SOX expression and a zoneof a high SOX11 expression, respectively (Fig. 8B), whereasexpression of SOX11 at a moderate level extended to the wideintermediate zone, which is externally positioned (Fig. 8Adand Bb and d).

On the other hand, the expression and subcellular localiza-tion of Brn2 changes dynamically between the ventricular andsubventricular zones and the intermediate zone. In the ven-tricular and subventricular zones, Brn2 expression was veryhigh, and the majority of Brn2 protein was localized in thenuclei of cells (Fig. 8Ca); in the intermediate zone, the expres-sion level was lower (Fig. 8Ae), and more significantly, theBrn2 protein was localized in the cytoplasm and excluded fromthe nucleus (Fig. 8Cb and c).

This regulation of the subcellular localization of Brn2 resultsin an interaction between Brn2 and SOX11 within the nucleusthat occurs only in the subventricular zone, in spite of the

FIG. 7. Expression of Nestin, group B1 SOX, SOX11, and Brn2 in E10.5 spinal cord. (A) Cross sections of E10.5 spinal cord at trunk levelimmunostained for Nestin (a), SOX2 (b), SOX1/(2)/3 (c), SOX11 (d), and Brn2 (e) and stained with DAPI (4�,6�-diamidino-2-phenylindole) (f).The scale bar indicates 100 �m. (B) High-magnification confocal images of median regions of the spinal cord (boxed in panel A) immunostainedfor SOX2 (a), SOX11 (b), SOX2 (green) and SOX11 (red) (c), SOX1/(2)/3 (d), Brn2 (e), and Brn2 (green) and Nestin (red) (f). The ventricle istoward the left. The scale bar indicates 50 �m.


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FIG. 8. Expression of Nestin, group B1 SOX, SOX11, and Brn2 in E13.5 spinal cord. (A) Cross sections of E13.5 spinal cord at trunk levelimmunostained for Nestin (a), SOX2 (b), SOX1/(2)/3 (c), SOX11 (d), Brn2 (e), and DAPI (4�,6�-diamidino-2-phenylindole) (f). The scale barindicates 100 �m. (B) High-magnification confocal images of median regions of the spinal cord roughly corresponding to the rectangles in panelA, which were immunostained for SOX2 (a), SOX11 (b), SOX1/(2)/3 (c), SOX2 (green) and SOX11 (red) (d), Brn2 (e), and Brn2 (green) andNestin (red) (f). The scale bar indicates 50 �m. The ventricle is toward the left. The ventricular zone (VZ), subventricular zone (SVZ), andintermediate zone (IZ) are indicated. Note that subcellular localization of Brn2 is clearly different in the VZ-SVZ and IZ. (C) Further enlargementof Brn2-immunostained specimens of the areas indicated in panel A, image e, which are also stained with DAPI (blue). In the ventricular zone(a), Brn2 signals are largely identified within the nucleus, although significant signals are also observed in the cytoplasm. In the nucleus, theheterochromatic regions strongly stained with DAPI are devoid of Brn2 signal. In the intermediate-zone areas (b and c), Brn2 signals are largelywithin the cytoplasm. The scale bar indicates 10 �m.

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expression of these transcription factors in the intermediatezone as well.

Thus, the zonal and subcellular distribution of group B1SOX, SOX11, and Brn2 supports the model that their syner-gism on the Nes30 core sequence is fully reflected in the acti-vation of the Nestin gene that takes place within the ventricularand subventricular zones.

Regulation of Brn2 activity by intracellular localization ofthe protein adds an important item to the list of cytoplasmicsequesterings of transcription factors in differentiated cells(e.g., N-myc in mature neurons [52] and SOX2 in trophoblasts[5]). Although we investigated only Brn2 among class III POUproteins in this study, a high degree of conservation of aminoacid sequences among the proteins of this class (1, 4, 18)supports the view that Brn1 and -4 and Oct6 are also under thesame regulation of subcellular localization.

Two populations of Nestin-expressing cells in the embryonicCNS. This study revealed that Nestin-expressing cells withinthe embryonic CNS are comprised of two major populations:(i) those expressing group B1 SOX and nuclear class III POU,located in the ventricular zone, and (ii) those expressingSOX11 (and possibly other group C SOX), together with nu-clear class III POU, located in the subventricular zone (Fig. 9).

The subventricular zone is a loosely defined zone of cellsthat are nonproliferative and positioned immediately externalto the ventricular zone. Previous in situ hybridization data ofthe spinal cord showed high Sox11 expression in the corre-sponding cell population (9). Based on the data presented inthis work, it may be logical to redefine the subventricular zone(at least in the embryonic spinal cord) as the zone character-ized by possession of nuclear class III POU proteins andSOX11. This argues that the shift of the cells from the ven-tricular zone to the subventricular zone indicates not only thechange of cell state from proliferative to nonproliferative (7,15, 37), but also the change of major transcriptional regulationfrom group B1 SOX dependent to group C SOX dependent.This change in the major SOX proteins may still maintainexpression of a significant fraction of genes, like Nestin, but

may cause altered expression of other genes; combinations ofSOX2-Brn2 and SOX11-Brn2 were both effective in activationof Nestin transcription through binding to Nes30 sequence(Fig. 6) but are also distinguished in other SOX-POU cobind-ing sites (26, 54).

Given that Nestin expression marks neural primordial cells,they are thus comprised of two populations: group B1 SOXdependent and group C SOX dependent (Fig. 9). In addition,class III POU proteins, the synergistic partners of the SOXproteins, are under the regulation of their subcellular localiza-tion. Nestin-expressing cells in later development and in theadult CNS remain to be investigated from this viewpoint, butregulation of embryonic Nestin expression must have an im-portant bearing on transcriptional regulation in later neuralprimordial cells.

ACKNOWLEDGMENTS

We thank members of the Kondoh laboratory for stimulating dis-cussions and Masato Nakafuku for the provision of MNS70 cells.

This work was supported by grants from the Ministry of Education,Culture, Sports, Science and Technology of Japan (12CE2007) andfrom the Human Frontier Science Program (RGP0040/2001-M) toH.K. and from the National Key Basic Research and DevelopmentalProgram (G1999054000 and 2002CB713802) and the National NaturalScience Foundation of China (39870283 and 39930090) to N.J.

REFERENCES

1. Alvarez-Bolado, G., M. G. Rosenfeld, and L. W. Swanson. 1995. Model offorebrain regionalization based on spatiotemporal patterns of POU-III ho-meobox gene expression, birthdates, and morphological features. J. Comp.Neurol. 355:237–295.

2. Alvarez-Buylla, A., B. Seri, and F. Doetsch. 2002. Identification of neuralstem cells in the adult vertebrate brain. Brain Res. Bull. 57:751–758.

3. Ambrosetti, D. C., C. Basilico, and L. Dailey. 1997. Synergistic activation ofthe fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends onprotein-protein interactions facilitated by a specific spatial arrangement offactor binding sites. Mol. Cell. Biol. 17:6321–6329.

4. Andersen, B., and M. G. Rosenfeld. 2001. POU domain factors in the neu-roendocrine system: lessons from developmental biology provide insightsinto human disease. Endocr. Rev. 22:2–35.

5. Avilion, A. A., S. K. Nicolis, L. H. Pevny, L. Perez, N. Vivian, and R.Lovell-Badge. 2003. Multipotent cell lineages in early mouse developmentdepend on SOX2 function. Genes Dev. 17:126–140.

6. Bowles, J., G. Schepers, and P. Koopman. 2000. Phylogeny of the SOXfamily of developmental transcription factors based on sequence and struc-tural indicators. Dev. Biol. 227:239–255.

7. Bylund, M., E. Andersson, B. G. Novitch, and J. Muhr. 2003. Vertebrateneurogenesis is counteracted by Sox1–3 activity. Nat. Neurosci. 6:1162–1168.

8. Cai, J., Y. Wu, T. Mirua, J. L. Pierce, M. T. Lucero, K. H. Albertine, G. J.Spangrude, and M. S. Rao. 2002. Properties of a fetal multipotent neuralstem cell (NEP cell). Dev. Biol. 251:221–240.

9. Cheung, M., M. Abu-Elmagd, H. Clevers, and P. J. Scotting. 2000. Roles ofSox4 in central nervous system development. Brain Res. Mol. Brain Res.79:180–191.

10. Cui, H., and R. F. Bulleit. 1998. Expression of the POU transcription factorBrn-5 is an early event in the terminal differentiation of CNS neurons.J. Neurosci. Res. 52:625–632.

11. Dahlstrand, J., M. Lardelli, and U. Lendahl. 1995. Nestin mRNA expressioncorrelates with the central nervous system progenitor cell state in many, butnot all, regions of developing central nervous system. Brain Res. Dev. BrainRes. 84:109–129.

12. Dailey, L., and C. Basilico. 2001. Coevolution of HMG domains and home-odomains and the generation of transcriptional regulation by Sox/POU com-plexes. J. Cell Physiol. 186:315–328.

13. Di Rocco, G., A. Gavalas, H. Popperl, R. Krumlauf, F. Mavilio, and V.Zappavigna. 2001. The recruitment of SOX/OCT complexes and the differ-ential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatoryenhancer function. J. Biol. Chem. 276:20506–20515.

14. Goto, K., T. S. Okada, and H. Kondoh. 1990. Functional cooperation oflens-specific and nonspecific elements in the delta 1-crystallin enhancer. Mol.Cell. Biol. 10:958–964.

15. Graham, V., J. Khudyakov, P. Ellis, and L. Pevny. 2003. SOX2 functions tomaintain neural progenitor identity. Neuron 39:749–765.

16. Hara, Y., A. C. Rovescalli, Y. Kim, and M. Nirenberg. 1992. Structure and

FIG. 9. Model of Nestin gene regulation in the embryonic spinalcord. Nestin is expressed in the cells with their cell bodies in theventricular zone (VZ) and subventricular zone (SVZ). In the ventric-ular zone, SOX1/2/3 (group B SOX) interact with Brn2 (and otherclass III POU factors) on the Nes30 sequence (Fig. 1 and 2) within thenucleus. In the subventricular zone, SOX11 (and possibly other groupC SOX, i.e., SOX4 and SOX12) interacts with class III POU factors. Inthe cells with cell body localization in the intermediate zone (IZ),moderate expression of SOX11 and Brn2 also takes place, but seques-tering the majority of Brn2 activity in the cytoplasm results in theabsence of Nestin expression in these cells.


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nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 11

Nov

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21 b

y 11

9.14

9.12

6.19

8.

evolution of four POU domain genes expressed in mouse brain. Proc. Natl.Acad. Sci. USA 89:3280–3284.

17. Hargrave, M., E. Wright, J. Kun, J. Emery, L. Cooper, and P. Koopman.1997. Expression of the Sox11 gene in mouse embryos suggests roles inneuronal maturation and epithelio-mesenchymal induction. Dev. Dyn. 210:79–86.

18. He, X., M. N. Treacy, D. M. Simmons, H. A. Ingraham, L. W. Swanson, andM. G. Rosenfeld. 1989. Expression of a large family of POU-domain regu-latory genes in mammalian brain development. Nature 340:35–41.

19. Herr, W., and M. A. Cleary. 1995. The POU domain: versatility in transcrip-tional regulation by a flexible two-in-one DNA-binding domain. Genes Dev.9:1679–1693.

20. Jay, P., I. Sahly, C. Goze, S. Taviaux, F. Poulat, G. Couly, M. Abitbol, andP. Berta. 1997. SOX22 is a new member of the SOX gene family, mainlyexpressed in human nervous tissue. Hum. Mol. Genet. 6:1069–1077.

21. Josephson, R., T. Muller, J. Pickel, S. Okabe, K. Reynolds, P. A. Turner, A.Zimmer, and R. D. McKay. 1998. POU transcription factors control expres-sion of CNS stem cell-specific genes. Development 125:3087–3100.

22. Kamachi, Y., K. S. Cheah, and H. Kondoh. 1999. Mechanism of regulatorytarget selection by the SOX high-mobility-group domain proteins as revealedby comparison of SOX1/2/3 and SOX9. Mol. Cell. Biol. 19:107–120.

23. Kamachi, Y., M. Uchikawa, J. Collignon, R. Lovell-Badge, and H. Kondoh.1998. Involvement of Sox1, 2 and 3 in the early and subsequent molecularevents of lens induction. Development 125:2521–2532.

24. Kamachi, Y., M. Uchikawa, and H. Kondoh. 2000. Pairing SOX off withpartners in the regulation of embryonic development. Trends Genet. 16:182–187.

25. Kamachi, Y., M. Uchikawa, A. Tanouchi, R. Sekido, and H. Kondoh. 2001.Pax6 and SOX2 form a co-DNA-binding partner complex that regulatesinitiation of lens development. Genes Dev. 15:1272–1286.

26. Kuhlbrodt, K., B. Herbarth, E. Sock, J. Enderich, I. Hermans-Borgmeyer,and M. Wegner. 1998. Cooperative function of POU proteins and SOXproteins in glial cells. J. Biol. Chem. 273:16050–16057.

27. Latchman, D. S. 1999. POU family transcription factors in the nervoussystem. J. Cell Physiol. 179:126–133.

28. Lendahl, U., L. B. Zimmerman, and R. D. McKay. 1990. CNS stem cellsexpress a new class of intermediate filament protein. Cell 60:585–595.

29. Lothian, C., and U. Lendahl. 1997. An evolutionarily conserved region in thesecond intron of the human nestin gene directs gene expression to CNSprogenitor cells and to early neural crest cells. Eur. J. Neurosci. 9:452–462.

30. McEvilly, R. J., M. O. de Diaz, M. D. Schonemann, F. Hooshmand, andM. G. Rosenfeld. 2002. Transcriptional regulation of cortical neuron migra-tion by POU domain factors. Science 295:1528–1532.

31. Mizuseki, K., M. Kishi, M. Matsui, S. Nakanishi, and Y. Sasai. 1998. Xe-nopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinctactivities in the initiation of neural induction. Development 125:579–587.

32. Nakagawa, Y., T. Kaneko, T. Ogura, T. Suzuki, M. Torii, K. Kaibuchi, K.Arai, S. Nakamura, and M. Nakafuku. 1996. Roles of cell-autonomousmechanisms for differential expression of region-specific transcription fac-tors in neuroepithelial cells. Development 122:2449–2464.

33. Nishimoto, M., A. Fukushima, A. Okuda, and M. Muramatsu. 1999. Thegene for the embryonic stem cell coactivator UTF1 carries a regulatoryelement which selectively interacts with a complex composed of Oct-3/4 andSox-2. Mol. Cell. Biol. 19:5453–5465.

34. Okamoto, K., H. Okazawa, A. Okuda, M. Sakai, M. Muramatsu, and H.Hamada. 1990. A novel octamer binding transcription factor is differentiallyexpressed in mouse embryonic cells. Cell 60:461–472.

35. Okamoto, K., M. Wakamiya, S. Noji, E. Koyama, S. Taniguchi, R. Take-mura, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, M. Muramatsu, et al.1993. A novel class of murine POU gene predominantly expressed in centralnervous system. J. Biol. Chem. 268:7449–7457.

36. Pevny, L. H., and R. Lovell-Badge. 1997. Sox genes find their feet. Curr.Opin. Genet. Dev. 7:338–344.

37. Pevny, L. H., S. Sockanathan, M. Placzek, and R. Lovell-Badge. 1998. A rolefor SOX1 in neural determination. Development 125:1967–1978.

38. Reim, G., and M. Brand. 2002. Spiel-ohne-grenzen/pou2 mediates regionalcompetence to respond to Fgf8 during zebrafish early neural development.Development 129:917–933.

39. Remenyi, A., K. Lins, L. J. Nissen, R. Reinbold, H. R. Scholer, and M.Wilmanns. 2003. Crystal structure of a POU/HMG/DNA ternary complexsuggests differential assembly of Oct4 and Sox2 on two enhancers. GenesDev. 17:2048–2059.

40. Rex, M., A. Orme, D. Uwanogho, K. Tointon, P. M. Wigmore, P. T. Sharpe,and P. J. Scotting. 1997. Dynamic expression of chicken Sox2 and Sox3 genesin ectoderm induced to form neural tissue. Dev. Dyn. 209:323–332.

41. Rietze, R. L., H. Valcanis, G. F. Brooker, T. Thomas, A. K. Voss, and P. F.Bartlett. 2001. Purification of a pluripotent neural stem cell from the adultmouse brain. Nature 412:736–739.

42. Scholer, H. R., A. K. Hatzopoulos, R. Balling, N. Suzuki, and P. Gruss. 1989.A family of octamer-specific proteins present during mouse embryogenesis:evidence for germline-specific expression of an Oct factor. EMBO J. 8:2543–2550.

43. Schwartz, P. H., P. J. Bryant, T. J. Fuja, H. Su, D. K. O’Dowd, and H.Klassen. 2003. Isolation and characterization of neural progenitor cells frompost-mortem human cortex. J. Neurosci. Res. 74:838–851.

44. Stolt, C. C., P. Lommes, E. Sock, M. C. Chaboissier, A. Schedl, and M.Wegner. 2003. The Sox9 transcription factor determines glial fate choice inthe developing spinal cord. Genes Dev. 17:1677–1689.

45. Streit, A., S. Sockanathan, L. Perez, M. Rex, P. J. Scotting, P. T. Sharpe, R.Lovell-Badge, and C. D. Stern. 1997. Preventing the loss of competence forneural induction: HGF/SF, L5 and Sox-2. Development 124:1191–1202.

46. Sugitani, Y., S. Nakai, O. Minowa, M. Nishi, K. Jishage, H. Kawano, K.Mori, M. Ogawa, and T. Noda. 2002. Brn-1 and Brn-2 share crucial roles inthe production and positioning of mouse neocortical neurons. Genes Dev.16:1760–1765.

47. Temple, S. 2001. The development of neural stem cells. Nature 414:112–117.48. Tomioka, M., M. Nishimoto, S. Miyagi, T. Katayanagi, N. Fukui, H. Niwa,

M. Muramatsu, and A. Okuda. 2002. Identification of Sox-2 regulatoryregion which is under the control of Oct-3/4-Sox-2 complex. Nucleic AcidsRes. 30:3202–3213.

49. Uchikawa, M., Y. Ishida, T. Takemoto, Y. Kamachi, and H. Kondoh. 2003.Functional analysis of chicken Sox2 enhancers highlights an array of diverseregulatory elements that are conserved in mammals. Dev. Cell. 4:509–519.

50. Uchikawa, M., Y. Kamachi, and H. Kondoh. 1999. Two distinct subgroups ofGroup B Sox genes for transcriptional activators and repressors: their ex-pression during embryonic organogenesis of the chicken. Mech. Dev. 84:103–120.

51. Uwanogho, D., M. Rex, E. J. Cartwright, G. Pearl, C. Healy, P. J. Scotting,and P. T. Sharpe. 1995. Embryonic expression of the chicken Sox2, Sox3 andSox11 genes suggests an interactive role in neuronal development. Mech.Dev. 49:23–36.

52. Wakamatsu, Y., Y. Watanabe, H. Nakamura, and H. Kondoh. 1997. Regu-lation of the neural crest cell fate by N-myc: promotion of ventral migrationand neuronal differentiation. Development 124:1953–1962.

53. Wegner, M. 1999. From head to toes: the multiple facets of Sox proteins.Nucleic Acids Res. 27:1409–1420.

54. Wiebe, M. S., T. K. Nowling, and A. Rizzino. 2003. Identification of noveldomains within Sox-2 and Sox-11 involved in autoinhibition of DNA bindingand partnership specificity. J. Biol. Chem. 278:17901–17911.

55. Williams, D. C., Jr., M. Cai, and G. M. Clore. 2004. Molecular basis forsynergistic transcriptional activation by Oct1 and Sox2 revealed from thesolution structure of the 42-kDa Oct1.Sox2.Hoxb1-DNA ternary transcrip-tion factor complex. J. Biol. Chem. 279:1449–1457.

56. Wilson, M., and P. Koopman. 2002. Matching SOX: partner proteins andco-factors of the SOX family of transcriptional regulators. Curr. Opin.Genet. Dev. 12:441–446.

57. Wood, H. B., and V. Episkopou. 1999. Comparative expression of the mouseSox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages.Mech. Dev. 86:197–201.

58. Yaworsky, P. J., and C. Kappen. 1999. Heterogeneity of neural progenitorcells revealed by enhancers in the nestin gene. Dev. Biol. 205:309–321.

59. Yuan, H., N. Corbi, C. Basilico, and L. Dailey. 1995. Developmental-specificactivity of the FGF-4 enhancer requires the synergistic action of Sox2 andOct-3. Genes Dev. 9:2635–2645.

60. Zappone, M. V., R. Galli, R. Catena, N. Meani, S. De Biasi, E. Mattei, C.Tiveron, A. L. Vescovi, R. Lovell-Badge, S. Ottolenghi, and S. K. Nicolis.2000. Sox2 regulatory sequences direct expression of a (beta)-geo transgeneto telencephalic neural stem cells and precursors of the mouse embryo,revealing regionalization of gene expression in CNS stem cells. Development127:2367–2382.

61. Zimmerman, L., B. Parr, U. Lendahl, M. Cunningham, R. McKay, B. Gavin,J. Mann, G. Vassileva, and A. McMahon. 1994. Independent regulatoryelements in the nestin gene direct transgene expression to neural stem cellsor muscle precursors. Neuron 12:11–24.


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