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Supporting InformationDanno et al. 10.1073/pnas.0710954105SI Materials and MethodsDNA Constructs. To generate the expression vector that containedsix repeats of the myc tag, pCS2–6xMyc, we digested theMyc-Xdsh-pCS2 vector with the restriction enzyme EcoRI. Thisvector contains two EcoRI recognition sites: one just upstreamof the start codon of Xdsh, and the other just downstream of thestop codon of Xdsh. Digestion with EcoRI excised the codingsequence of Xdsh, and the subsequent self-ligation generated thepCS2–6xMyc vector. Similarly, to generate the expression vectorthat contained three repeats of the FLAG tag, pCS2–3xFLAG,we digested the pCS-Cripto-3FLAG vector with NcoI. Thisvector contains two NcoI recognition sites: one just upstream ofthe start codon of Cripto, and the other just downstream of thestop codon of Cripto. Digestion with NcoI excised the codingsequence of Cripto, and the subsequent self-ligation generatedthe pCS2–3xFlag vector.
The coding region of Xenopus laevis Otx2 was amplified byPCR using as template the XBm-Xotx2 plasmid, which was a giftfrom Maria Pannese (Istituto Scientifico H San Raffaele, Milan,Italy), and the following primers: forward, 5�-cATGATGTCT-TATCTCAAGCAACC-3� and reverse, 5�-TCACAAAACCT-GGAACTTCC-3�. The Otx2 fragment was inserted intoEcoRV-digested pCS2–6xMyc, to generate pCS2-myc-XOtx2.Because the EcoRV recognition site is located just downstreamof the Myc tags in the pCS2–6xMyc vector, pCS2-myc-XOtx2encodes N-terminally myc-tagged XOtx2. Similarly, the PCR-amplified Otx2 fragment was inserted into EcoRV-digestedpBluescript SK-, to generate XOtx2-SK. To generate the pCS2-FLAG-XOtx2 construct that encoded N-terminally FLAG-tagged XOtx2, we digested XOtx2-SK with EcoRI and XhoI andinserted this fragment into pCS2–3xFLAG that was digestedwith EcoRI and XhoI.
The pCS2-XSox2 plasmid and the pCS2-HA-XSox2 plasmid,which contains C-terminally HA-tagged X. laevis Sox2 (Gen-Bank accession no. BC076717), were gifts from Kazuyoshi R.Nitta (Institut de Biologie du Développement de MarseilleLuminy, Marseille, France). To generate the pCS2-myc-Sox2construct that encodes N-terminally myc-tagged Sox2, we am-plified by PCR the coding region of Sox2 using the pCS2-HA-XSox2 plasmid as a template and the following two primers:forward, 5�-cATGTACAGCATGATGGAGAC-3� and reverse,5�-TCACATGTGCGACAGAGGCAG-3�. This PCR fragmentwas inserted into EcoRV-digested pCS2–6xMyc. The dominant-negative Sox2 construct, pCS2-dn-XSox2, was generated accord-ing to the construction strategy described for Sox2BD(-) in aprevious report (1). Briefly, the Sox2-coding region lacking mostof the high-mobility group (HMG) domain (amino acids 105–355) was inserted into the pCS2 vector, which contains a nuclearlocalization signal.
To generate the pCS2-HA-XTcf-3 vector, which encodesN-terminally HA-tagged X. laevis Tcf-3, we amplified by PCRthe HA-XTcf-3 fragments using the pcDNAXTcf-3 plasmid astemplate and two linker primers that contain XhoI and XbaIsites. The pcDNAXTcf-3 vector was a gift from Hans Clevers(Hubrecht Institute, Utrecht, The Netherlands). The PCR frag-ment was inserted into pCS2 that was digested with XhoI andXbaI.
The point mutations in Otx2 (pCS2-myc-XOtx2-K50Q, pCS2-myc-XOtx2-K50A, and pCS2-myc-XOtx2-K50E) and Sox2(pCS2-HA-XSox2-R74P and pCS2-HA-XSox2-L97P) were gen-erated by inverse PCR with the following primers: for K50Q,forward, 5�-CAGAACCGCAGAGCAAAGTGC-3� and re-
verse, 5�-GAACCAGACCTGGACTCTGG-3�; for K50A, for-ward, 5�-GAGAACCGCAGAGCAAAGTGC-3� and reverse,5�-GAACCAGACCTGGACTCTGG-3�; for K50E, forward, 5�-GCCAACCGCAGAGCAAAGTGC-3� and reverse, 5�-GAACCAGACCTGGACTCTGG-3�; for R74P, forward, 5�-ACTGGGGGCTGAGTGGAAG-3� and reverse, 5�-GGC-TTGCTGATCTCCGAGTTG-3�; and for L97P, forward, 5�-CACGAGCTCTGCACATGAAG-3� and reverse, 5�-GC-CTCTTGGCCTCGTCGATG-3�.
To generate the GST fusion with Sox2, pCS2-XSox2 wasdigested with EcoRI and XhoI, and the Sox2 fragment wasinserted into pGEX-4T-1 (GE Healthcare) digested with EcoRIand XhoI. Similarly, to generate the GST fusion with Otx2,XOtx2-SK was digested with EcoRI and XhoI, and the Otx2fragment was inserted into pGEX-4T-3 (GE Healthcare) di-gested with EcoRI and XhoI. To generate the deleted form ofthe GST fusion with Sox2 or Otx2, we performed PCR usingXSox2-SK or XOtx2-SK as the template and the T3, T7, or linkerprimers. The PCR fragments were digested with EcoRI andXhoI and inserted into EcoRI- and XhoI-digested pGEX-4T-3.The amino acid positions (in parentheses) and the primers usedfor the deletion constructs of X. laevis Sox2 and Otx2 were asfollows: for Sox2-N (1–134), forward, T3 primer and reverse,5�-gggctcgagGGCGTTGGCCCCAGGAGCCAGC-3�; forSox2-HMG (35–114), forward, 5�-ggggaattcCGACCGAGT-GAAGAGACCCATG-3� and reverse, 5�-gggctcgagGGTTTT-GGTTTTCCTCCTGGG-3�; for Sox2-C (135–311), forward,5�-ggggaattcCATGACTTCTGGGGTCGGGGGC-3� and re-verse, T7 primer; for Sox2-HMG-��1 (64–114), forward, 5�-ggggaattcCAACTCGGAGATCAGCAAGAG-3� and reverse,5�-gggctcgagGGTTTTGGTTTTCCTCCTGGG-3�; for Sox2-HMG-�tail (35–99), forward, 5�-ggggaattcCGACCGAGT-GAAGAGACCCATG-3� and reverse, 5�-gggctcgagCTTCAT-GTGCAGAGCTCGCAG-3�; for Sox2-HMG-tail (95–114),forward, 5�-ggggaattcCGCTCTGCACATGAAGGAGC-ATC-3� and reverse, 5�-gggctcgagGGTTTTGGTTTTCCTC-CTGGG-3�; for Otx2-N (1–68), forward, T3 primer and reverse,5�-gggctcgagTCTCATGAAGATATCAGGG-3�; for Otx2-HD-1 (28–110) forward, 5�-ggggaattcCTCGGTGGGATATC-CCGCGAC-3� and reverse, 5�-gggctcgagAGGTCTCACTTT-GTTTTGGCCTC-3�; for Otx2-HD-2 (28–100), forward, 5�-ggggaattcCTCGGTGGGATATCCCGCGAC-3� and reverse,5�-gggctcgagCTGCTGCTGTTGCTGCTGCCGGC-3�; forOtx2-HD-3 (38–110), forward, 5�-ggggaattcCCAGAGGAGG-GAAAGGACCAC-3� and reverse, 5�-gggctcgagAGGTCT-CACTTTGTTTTGGCCTC-3�; for Otx2-HD-4 (38–100), for-ward, 5�-ggggaattcCCAGAGGAGGGAAAGGACCAC-3� andreverse, 5�-gggctcgagCTGCTGCTGTTGCTGCTGCCGGC-3�;for Otx2-M1 (101–213), forward, 5�-ggggaattcCCAGAATG-GAGGCCAAAAC-3� and reverse, 5�-gggctcgagATAGAGTT-GATGATGCATAGG-3�; for Otx2-M2 (160–233), forward, 5�-ggggaattcCCTGTCCGACCCCCTGTCCAC-3� and reverse, 5�-gggctcgagTCCCCACTGGTTAAGGTGAC-3�; and for Otx2-C(214 –288), forward, 5�-ggggaattcCGGACCTGGAGCTA-CTCTAAGC-3� and reverse, T7 primer.
For the isolation of a X. laevis genomic clone that contains theregion upstream of Rax, three forward primers and three reverseprimers were designed according to the sequence in the database(GenBank accession no. AY250711). PCR was performed byusing a liver-derived genome library of X. laevis as the templateand these primers. However, a Rax-specific fragment was am-plified for only one combination of primers (forward, 5�-
Danno et al. www.pnas.org/cgi/content/short/0710954105 1 of 10
CTGCTCACAAACTCCAAACACTTGTC-3� and reverse,5�-CTGCAGGGGCAGGATGGTATATTCCAC-3�). The am-plified fragment, which was named RaxG4 (GenBankAB365789), was inserted into pBluescript SK� digested withEcoRV, to generate RaxG4-SK. To generate the EGFP reportervector pRax-2600b-EGFP, RaxG4-SK was digested with XhoIand BamHI, and the RaxG4 fragment was inserted intopd2EGFP-1 (Clontech) that was digested with XhoI and BamHI.To generate the luciferase reporter vector pRax-2600b-Luc,pRax-2600b-EGFP was digested with XhoI and NcoI, and theRaxG4 fragment was inserted into pGL3-Basic (Promega) thatwas digested with XhoI and NcoI. The 5�-deleted constructs forthe Rax upstream region were generated as follows. The pRax-2600b-EGFP was digested with XhoI and SacI or XhoI and XbaIand blunted with T4 DNA Polymerase (Takara), followed byself-ligation to generate pRax-2300b-EGFP or pRax-1870b-EGFP, respectively. The pRax-2600b-Luc plasmid was digestedwith SacI, SacI and ApaI, or XhoI and BglII and blunt-endedwith T4 DNA polymerase followed by self-ligation to generatepRax-2300b-Luc, pRax-2180b-Luc, or pRax-1740b-Luc, respec-tively.
Tandem repeat reporter vectors (SOP-FLASH, mS-FLASH,and mO-FLASH) were constructed as follows. The sense andantisense oligonucleotides for pentatriacontamer (pt), mS-pt, ormO-pt were mixed and phosphorylated with T4 polynucleotidekinase (Takara) at 37°C for 1 h. The DNA sequences of theseoligonucleotides were as follows: pt sense, 5�-GTAGATTATC-TACTAACAATGGGCCCCTGGCTGAG-3�; pt antisense, 5�-CTCAGCCAGGGGCCCATTGTTAGTAGATAATCTAC-3�;mS-pt sense, 5�-GTAGATTATCTACTAACgcTGGGCCCCT-GGCTGAG-3�; mS-pt antisense, 5�-CTCAGCCAGGGGC-CCAgcGTTAGTAGATAATCTAC-3�; mO-pt sense, 5�-GTAGAgcATCTACTAACAATGGGCCCCTGGCTGAG-3�;and mO-pt antisense, 5�-CTCAGCCAGGGGCCCATTGT-TAGTAGATgcTCTAC-3�. Substituted nucleotides are indi-cated in lowercase. These oligonucleotides were also used in theEMSA as probes or competitors. To anneal the sense andantisense oligonucleotides, the mixture was heated at 95°C for10 s in a block incubator, and the incubator was then turned off.After the temperature of the block cooled to 25°C, an equalvolume of DNA ligation mix (Takara) was added to each tubeand incubated at 16°C for 2 h. The ligation products wereelectrophoresed in a 3% agarose gel. Gel bands of �200 bp wereextracted and purified with the MinElute gel extraction kit(Qiagen). The fragments were again phosphorylated by T4polynucleotide kinase at 37°C for 1 h and subjected to ligationwith EcoRV-digested pBluescript SK�, generating SOP-SK,mS-SK, or mO-SK. These constructs were verified by DNAsequencing. Eight tandem repeats of pt or mS-pt were excisedfrom SOP-SK or mS-SK by using KpnI and BamHI and insertedinto pGL3-Promoter (Promega) digested with KpnI and BamHIto generate SOP-FLASH or mS-FLASH, respectively. To gen-erate mO-FLASH, seven tandem repeats of mO-pt were excisedfrom mO-SK by using SacI and XhoI and inserted into pGL3-Promoter digested with SacI and XhoI.
RT-PCR and 5�-RACE. X. laevis embryos or animal cap explants werecultured to the late neurula stage and harvested. Total RNA wasextracted from two whole embryos or 15 animal cap explants byusing ISOGEN (Wako). The cDNA was synthesized from 800 ngof total RNA by using SuperScript II reverse transcriptase(Invitrogen) and oligo(dT)12–18 primer (Invitrogen) and sub-jected to PCR analysis. The primers, annealing temperature, andnumber of cycles for each gene were as follows: for ODC,5�-GCCATTGTGAAGACTCTCTCCATTC-3� and 5�-TTCGGGTGATTCCTTGCCAC-3�, 56°C, 28 cycles; for Rax,5�-CCCTATGGAGATCCATATTCAGG-3� and 5�-CTCT-TCTCTGCTGTATACGTCGG-3�, 56°C, 30 cycles; for Sox2,
5�-GAGGATGGACACTTATGCCCAC-3� and 5�-GGACAT-GCTGTAGGTAGGCGA-3�, 56°C, 28 cycles; and for ms-actin,5�-TTGCTTGGAGGAGTGTGT-3� and 5�-GCTGACA-GAATGCAGAAG-3�, 56°C, 28 cycles. 5�-RACE was per-formed using the First Choice RLM-RACE kit (Ambion). TotalRNA was extracted from the anterior neuroectoderm of stage-13embryos and subjected to 5�-RACE. The Rax-specific RACEprimers were as follows: outer, 5�-TCCTGAGCAGATGTCCA-GACAGAGAG-3� and inner, 5�-CGCGGATCCGAACACT-GCGTTTGCTGGCTTTGATG-3�.
ChIP Assay. For ChIP of endogenous proteins, headpieces weredissected from 500 embryos at stages 20–23. The headpieceswere washed with PBS and cross-linked with 1% formaldehydein PBS for 20 min at room temperature. To stop cross-linking,headpieces were treated with 0.125 M glycine for 10 min at roomtemperature and then washed twice with PBS. After gentlewashing with ChIP lysis buffer [1% SDS, 10 mM EDTA, 50 mMTris�HCl (pH 8.0), Complete protease inhibitor mixture(Roche)], the headpieces were lysed with 200 �l of ChIP lysisbuffer with pipetting. The lysates were then mixed with 700 �lof ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mMEDTA, 16.7 mM Tris�HCl (pH 8.0), 167 mM NaCl, Completeprotease inhibitor mixture] and sonicated six times for 10 s eachat the maximum setting (Sonifier 150; Branson). Then, 1 ml ofChIP dilution buffer was added, and the samples were centri-fuged at 17,000 � g for 10 min. The supernatant was transferredinto a fresh centrifuge tube and recentrifuged twice under thesame conditions, to remove the yolk. An aliquot of 100 �l of thesupernatant was stored at 4°C as the input DNA sample. Theremainder of the supernatant was precleared by incubation for1 h at 4°C with 15 �g of normal rabbit IgG (Upstate Biotech-nology) and 75 �l of protein G–agarose/salmon sperm DNA(Upstate Biotechnology). The supernatant was collected bycentrifugation at 6,000 � g for 10 s and divided into three freshcentrifuge tubes. To each lysate was added normal rabbit IgG,anti-Otx2 antibody (ab21990; Abcam), or anti-Sox2 antibody(Y-17; Santa Cruz Biotechnology), and the samples were incu-bated on a rotator at 4°C for 8–12 h. After the addition of 25 �lof protein G–agarose/salmon sperm DNA, the samples wereincubated at 4°C for 1 h. Precipitates were washed sequentiallyfor 5 min each in low-salt buffer [0.1% SDS, 1% Triton X-100,2 mM EDTA, 20 mM Tris�HCl (pH 8.0), 150 mM NaCl],high-salt buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris�HCl (pH 8.0), 500 mM NaCl], and LiCl buffer [0.25 MLiCl, 1% Nonidet P-40, 1% deoxycholic acid, 1 mM EDTA, 10mM Tris�HCl (pH 8.0)]. The precipitates were then washed twicefor 5 min with ChIP TE buffer [10 mM Tris�HCl (pH 8.0), 1 mMEDTA]. The immunocomplex was extracted twice by incubationfor 15 min at room temperature with 50 �l of ChIP elution buffer(1% SDS, 0.1 M NaHCO3, 10 mM DTT). The eluates and inputDNA were supplemented with 5 M NaCl to a final concentrationof 200 mM and heated at 65°C for 8–12 h to reverse theformaldehyde cross-linking. The samples were sequentiallytreated each for 1 h with RNase I at 37°C and proteinase K at55°C. DNA fragments were purified by using the QIAquick PCRpurification Kit (Qiagen) and analyzed by PCR. The primers,annealing temperature, and number of cycles in the PCR analysiswere as follows: for CNS1, upper, 5�-TCATTAGGGAGCAT-TGTCATTC-3� and lower, 5�-CCAAAGGGGTAGTAAAGC-CAC-3�, 58°C, 32 cycles; for exon 2, upper, 5�-AAAGACCT-CAAGCGAGTGCCTG-3� and lower, 5�-TTCATAGC-CAGCTCTTCTCTGC-3�, 58°C, 32 cycles; and for exon 3,upper, 5�-AAGCAAAGGAACACATCCAG-3� and lower, 5�-AACACTGGCAAACTCCACAG-3�, 58°C, 32 cycles. For theChIP assay of overexpressed proteins, 50 embryos were injectedwith 100 ng of myc-Otx2 or myc-Sox2 mRNA at the eight-cellstage and cultured to the midblastula stage. Whole embryos were
Danno et al. www.pnas.org/cgi/content/short/0710954105 2 of 10
subjected to formaldehyde cross-linking and further ChIP pro-cedures. For immunoprecipitation, 5 �g of anti-myc antibody(9E10; Santa Cruz Biotechnology) was used.
EMSA. The proteins used in the EMSA were transcribed andtranslated from the expression vectors pCS2-myc-XOtx2, pCS2-HA-XSox2, and pCS2-HA-XTcf-3 by using the SP6 TnT coupledreticulocyte lysate system (Promega). The expression of theseproteins was confirmed by Western blotting (data not shown).The probes were prepared as follows. For the EMSA with aradioisotope, the sense and antisense oligonucleotides for ptwere radiolabeled by incubation with [�-32P]ATP (GE Health-care) and T4 polynucleotide kinase at 37°C for 1 h. Theradioisotope-labeled probe was then purified with the DyeEx 2.0spin kit (Qiagen) and annealed. For the EMSA without aradioisotope, the sense oligonucleotide for pt and the antisenseoligonucleotide for pt, which was labeled with Cy5.5 (Sigma–Aldrich), were mixed and annealed. The DNA—protein-bindingreaction was performed in binding buffer [20 mM Hepes (pH7.8), 45 mM KCl, 10 mM NaCl, 1 mM EDTA, 10% (vol/vol)glycerol, 0.1% Nonidet P-40, 0.2 mg/ml BSA, 1 mM DTT] atroom temperature for 30 min. After electrophoresis, the bindingreactions were analyzed by using BAS-5000 (Fujifilm) for theradioisotope-labeled probe or the Odyssey image reader (Li-Cor) for the Cy5.5-labeled probe. The sense strand sequences ofthe probes or competitors used in EMSA were as follows: for pt,5�-GTAGATTATCTACTAACAATGGGCCCCTGGCT-GAG-3�; for TCE, 5�-CCCAGAGCTTCAAAGGGTGC-CCTACTTG-3�; for mO-pt, 5�-GTAGAgcATCTACTAA-CAATGGGCCCCTGGCTGAG-3�; and for mS-pt, 5�-GTA-GATTATCTACTAACgcTGGGCCCCTGGCTGAG-3�.Substituted nucleotides are indicated in lowercase.
Immunocytochemistry and Confocal Microscopy. HEK293T cellsgrown in glass chambers coated with collagen type IA (cellmatrix) were transfected with expression vectors (pCS2-myc-XOtx2 and pCS2-HA-XSox2). Thirty hours after transfection,the cells were fixed, and standard immunofluorescence proce-dures were performed. The cells were analyzed by epifluores-cence or confocal microscopy. The following antibody sets wereused for immunocytochemistry: mouse anti-myc (9E10; SantaCruz Biotechnology) and donkey anti-mouse IgG-Alexa Fluor594 (A21203; Molecular Probes), and rabbit anti-HA (Y-11;Santa Cruz Biotechnology) and donkey anti-rabbit IgG-AlexaFluor 488 (A21206; Molecular Probes).
Coimmunoprecipitation (CoIP) Assay. HEK293T cells were grown ina 10-cm dish and transfected with 6 �g of pCS2-myc-XOtx2and/or 6 �g of pCS2-HA-XSox2 by using Lipofectamine 2000(Invitrogen). The total amounts of DNA were kept constant bythe addition of pCS2-nls-�gal, when needed. Thirty-six hoursafter transfection, the cells were washed twice with cold PBS andharvested with a scraper. Cells were transferred into a freshcentrifuge tube and washed twice in cold PBS by resuspensionand centrifugation. The final packed cell volume was 50 �l. Weperformed the two-step lysis method according to Klenova et al.(2). Cells were resuspended in 1 ml of CoIP lysis buffer [20 mMTris�HCl (pH 7.4), 10 mM KCl, 10 mM MgCl2, 2 mM EDTA,
10% (vol/vol) glycerol, 1% Triton X-100, 2.5 mM �-glycerophos-phate, 1 mM NaF, 1 mM DTT, Complete protease inhibitormixture] by pipetting and then incubated on ice for 10 min. Aftertwo rounds of sonication (Sonifier 150 at 20% output, 50% dutycycle, 10 s), 97 �l of 5 M NaCl (final concentration, 420 mM) wasadded. After incubation on ice for 90 min, the lysate wasresonicated under the same conditions. The lysate was centri-fuged at 17,000 � g for 25 min at 4°C, and the supernatant wastransferred into a new tube. For preclearing, the supernatant wassupplemented with 5 �g of normal mouse or rabbit IgG and 25�l of protein G–Sepharose 4 Fast Flow (GE Healthcare) andincubated at 4°C for 1 h on a rotary incubator. The supernatantwas recovered by centrifugation at 6,000 � g for 5 s at 4°C. Tothe supernatant was added 5 �g of anti-myc (9E10; Santa CruzBiotechnology) or 5 �g of anti-HA (Y-11; Santa Cruz Biotech-nology) antibody, and the mixture was incubated for 4–12 h at4°C on a rotary incubator. Then, 25 �l of protein G–Sepharose4 Fast Flow was added and incubated for an additional 2 h. Thebeads were washed five times by resuspending in CoIP lysisbuffer supplemented with NaCl to a final concentration of 200mM, followed by centrifugation at 6,000 � g for 5 s at 4°C. Tothe beads were added 25 �l of 2� SDS sample buffer [0.125 MTris�HCl (pH 6.8), 4% SDS, 40% (vol/vol) glycerol, 0.01%bromophenol blue, 100 mM DTT], and the samples were elutedby incubation for 3 min at 95°C. The eluted proteins wereanalyzed by SDS/PAGE and Western blotting.
GST Pulldown Assay and Quantification of Binding Levels. GST fusionproteins were extracted from Escherichia coli BL21 transfectedwith expression vectors. The cells that expressed GST fusionproteins were collected by centrifugation and lysed with GSTlysis buffer (PBS supplemented with 100 mM NaCl, 0.5% TritonX-100, and Complete protease inhibitor mixture), after sonica-tion (Sonifier 150, at 10% output, 50% duty cycle, 10 s). Themyc-, HA-, or FLAG-tagged proteins were synthesized by usingthe SP6 TnT coupled reticulocyte lysate system (Promega). GSTfusion proteins (10 �g) were bound to 25 �l of glutathione–Sepharose 4 Fast Flow (GE Healthcare) by incubation at 4°C for1 h. After three washes with GST lysis buffer, tagged proteinswere added to the bead-bound GST fusion proteins. The reac-tion was performed at 4°C for 2 h, and the precipitates werewashed five times with GST lysis buffer. The beads were mixedwith 25 �l of 2� SDS sample buffer, and the proteins were elutedby incubation at 95°C for 3 min and then analyzed by SDS/PAGEand Western blotting. The amounts of input and GST pulled-down Sox2 proteins were analyzed by Western blotting with theanti-HA (Y-11) and anti-rabbit IgG-IRDye 800CW antibodiesand detected using the Odyssey image reader (Li-Cor). TheOtx2-binding Sox2 to input Sox2 ratio of Sox2-WT is set at 100%,and the ratios obtained for the mutated Sox2 are expressed aspercentages relative to the control.
Western Blotting. The following antibodies were used for Westernblotting: anti-myc (A-14, Ab-1, and 9E10; Santa Cruz Biotech-nology), anti-HA (Y-11 and F-7; Santa Cruz Biotechnology),anti-HA-horseradish peroxidase (3F10; Roche), anti-FLAG(M2; Sigma), anti-Otx2 (ab21990; Abcam), anti-Sox2 (Y-17;Santa Cruz Biotechnology), anti-�-tubulin (T9026; Sigma), andanti-rabbit IgG-IRDye 800CW (611-131-003; Rockland).
1. Kishi M, et al. (2000) Requirement of Sox2-mediated signaling for differentiation ofearly Xenopus neuroectoderm. Development 127:791–800.
2. Klenova E, Chernukhin I, Inoue T, Shamsuddin S, Norton J (2002) Immunoprecipitationtechniques for the analysis of transcription factor complexes. Methods 26:254–259.
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Otx2
EGFP Merge
Sox2 Rax
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Fig. S1. Gene expression patterns in X. laevis embryos. (A) EGFP expression in a transgenic Xenopus embryo. The 2,600-nucleotide sequence upstream of theX. laevis Rax gene drives the expression of the reporter gene EGFP. At the neurula stage, EGFP protein is expressed in the optic vesicle, where endogenous Raxis expressed. The EGFP expression observed in the most-anterior neural tube is probably a remnant of earlier expression. (B) Comparison of the expression patternsof Otx2, Sox2, and Rax mRNA in X. laevis embryos at the late neurula stages (stages 22, 25, and 28). These genes are coexpressed in the eye primordia throughlate neural development.
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pt Otx2
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Fig. S2. EMSA and ChIP experiments demonstrating DNA binding by the Otx2 and Sox2 proteins. (A) Oligonucleotides used in the EMSA. The substitutednucleotides in mO-pt and mS-pt are indicated in lowercase and are underlined. (B) EMSA showing specific binding of Otx2 to CNS1 in vitro. The band shiftedby the Otx2 protein is decreased 100-fold by the addition of intact pt competitor but not by the addition of mO-pt. (C) EMSA showing that Sox2 binds specificallyto CNS1 in vitro. The specificity of the binding is demonstrated by competition with increasing amounts (from 100-fold to 1,000-fold excess) of pt or mS-pt. (D)The HMG class protein XTcf-3 does not bind to CNS1. XTcf-3 binds to the TCE oligonucleotide. (E) Experimental diagram showing ChIP after microinjection. (F)The ChIP assay demonstrates that overexpressed myc-Otx2 and myc-Sox2 proteins bind to Rax CNS1 in vivo. CNS1 fragments are immunoprecipitated by theanti-myc antibody but not by normal mouse IgG, from embryos injected with myc-Otx2 or myc-Sox2 mRNA. Other regions of the same chromosome as CNS1,i.e., exons 2 and 3, are immunoprecipitated to much lesser extents. (G) EMSA showing that mutation of the Sox-binding site does not affect the binding of Otx2to the Otx-binding site. The arrow indicates a band that is shifted by the binding of Otx2 to CNS1. This binding is decreased by the addition of pt or mS-ptcompetitors, whereas the addition of mO-pt competitors decreases the binding to a markedly lesser extent. Competitors were added at 10-, 100-, and 1,000-foldexcesses. (H) Quantification of the shifted band in G. A similar degree of competition is found with the addition of pt and mS-pt. (I) EMSA showing that themutated Sox2 proteins lose DNA-binding activity. Oligonucleotide pt, which is derived from Rax CNS1, was used as the probe.
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IB: Sox2
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IB: α -tubulin
A B
Fig. S3. Lack of endogenous expression of the Otx2 and Sox2 proteins in HEK293T cells. (A) In HEK293T cells, the Otx2 protein is not expressed or induced bytransfection with Sox2. HEK293T cells transfected with myc-Otx2 are used as a positive control. The arrow and arrowhead indicate endogenous Otx2 andexogenous myc-Otx2, respectively. (B) In HEK293T cells, the Sox2 protein is not expressed or induced by transfection with Otx2. Murine ES cells are used as apositive control.
Danno et al. www.pnas.org/cgi/content/short/0710954105 6 of 10
50
0
10
20
30
40
50
0
10
20
30
40
Sox2 Otx2 WT
- + + + + +
y t i v i t c a e s a r e f i c u l e v i t a l e R
HEK293T
KQ KA KE - -
Otx2
y t i v i t c a e s a r e f i c u l e v i t a l e R
mS-FLASH
SOP-FLASH
WT KQ KA KE -
B A
HEK293T
Fig. S4. Binding specificity of the Otx2 protein for DNA is required for the transactivation caused by Otx2. (A) Substitution of lysine 50 in the homeodomainof Otx2 with glutamine (KQ), alanine (KA), or glutamic acid (KE) abolishes the synergistic activation by Sox2 and Otx2 of the reporter vector SOP-FLASH. Theamounts of Otx2 (WT, KQ, KA, or KE) and Sox2 plasmids used were 100 ng and 30 ng, respectively. (B) For mS-FLASH, the increase in transcription caused by Otx2alone depends on the binding specificity of Otx2. The amount of Otx2 (WT, KQ, KA, or KE) plasmid used was 100 ng.
Danno et al. www.pnas.org/cgi/content/short/0710954105 7 of 10
T
S
G
G
M
H
- 2 x o S
-
T
S
G
- G
M
H
- 2 x o
S
- T
S
G
α ∆
1
- G
M
H
- 2 x o
S
- T
S
G
∆
l i a t l i a t - G
M
H
- 2 x o
S
- T
S
G
IB: myc
Coomassie
+ myc-Otx2
α 1 α 2 α 3 tail Sox2-HMG
Sox2-HMG- ∆α 1
Sox2-HMG- ∆ tail
Sox2-HMG-tail
Otx2 binding
+
+
+
-
Otx2-HD-1
Otx2-HD-2
Otx2-HD-3
Otx2-HD-4
homeodomain
+ HA-Sox2
t u p n I
T
S
G
T
W
- 2 x t O
-
T
S
G
1 - D
H
- 2 x t
O
- T
S
G
2 - D
H
- 2 x t
O
- T
S
G
3 - D
H
- 2 x t
O
- T
S
G
4 - D
H
- 2 x t
O
- T
S
G
Coomassie
IB: HA
E F
SVGYPATPRK
QRRERTTFTRAQLDILEALFA
KTRYPDIFMREEVALKINLPE
SRVQVWFKNRRAKCRQQQQQQ
QNGGQNKVRP
homeodomain
G H I
++++
++
++
+
Sox2 binding
α 1 α 2 α 3 tail
frog chicken mouse human
HMG domain of Sox2
α 1 α 2 α 3
human mouse chicken frog
Homeodomain of Otx2
J
K
L HA-Sox2 myc-Otx2 DAPI Merge
homeodomain WSP
homology Otx tails
Otx2-WT + Otx2-N + Otx2-HD-1 + Otx2-HD-4 -/+ Otx2-M1 - Otx2-M2 - Otx2-C -
Sox2 binding
IB: HA
Coomassie
T
S
G
T
W
- 2 x t O
- T
S
G
N
- 2 x t O
- T
S
G
1 - D
H
- 2 x t
O
- T
S
G
4 - D
H
- 2 x t
O
- T
S
G
1 M
- 2 x t
O
- T
S
G
2 M
- 2 x t
O
- T
S
G
C
- 2 x t O
- T
S
G
t u p n I
+ HA-Sox2 C D
Otx2 binding
HMG Sox2-WT +
Sox2-N +
Sox2-HMG +
Sox2-C -
T
S
G
t u p n I
T
W
- 2 x o S
-
T
S
G
N
- 2 x o S
-
T
S
G
G
M
H
- 2 x o S
-
T
S
G
C
- 2 x o S
-
T
S
G
IB: Flag
Coomassie
+ Flag-Otx2
A B
myc-Otx2 HA-Sox2 Merge M
Otx2-WT
Fig. S5. Characterization of the interaction between the Otx2 and Sox2 proteins. (A) Depiction of the constructs used in B. (B) FLAG-Otx2 was subjected topulldown by GST/GST-Sox2 (-WT, -N, -HMG, or -C). Binding to Otx2 is indicated as � (positive) or � (negative). (C) Depiction of the constructs used in D. (D) HA-Sox2was subjected to pulldown by GST/GST-Otx2 (-WT, -N, -HD-1, -HD-4, -M1, -M2, or -C). (E) Depiction of the constructs used in F. (F) Myc-Otx2 was subjected topulldown by GST/GST-Sox2 (-HMG, -HMG-��1, -HMG-�tail, or -HMG-tail). The results suggest that helices 2 and 3 of the HMG domain are essential for the bindingbetween Sox2 and Otx2. (G) Amino acid sequences of the homeodomain of Otx2 and its flanking residues. The homeodomain is indicated as a square. (H)Depiction of the constructs used in I. (I) HA-Sox2 was subjected to pulldown by GST/GST-Otx2 (-WT, -HD-1, -HD-2, -HD-3, or -HD-4). The N- and C-flanking residuesof the homeodomain modulate the interaction between Otx2 and Sox2. (J) Conservation of the HMG domain of the Sox2 protein. Black boxes indicate the threehelices that form the HMG domain. (K) Conservation of the homeodomain and its adjacent residues in the Otx2 protein. Black boxes indicate the three helicesthat form the homeodomain. (L) Immunocytochemistry showing that HA-Sox2 (green) and myc-Otx2 (red) colocalize to the nucleus (DAPI, blue). HEK293T cellswere transfected with HA-Sox2 and myc-Otx2 and immunostained with anti-HA and anti-myc antibodies. (M) Cells that coexpressed HA-Sox2 and myc-Otx2 werefurther analyzed by confocal microscopy.
Danno et al. www.pnas.org/cgi/content/short/0710954105 8 of 10
Sox2 Otx2
Sox site Otx site
Rax
Otx2
Sox site Otx site
Rax
Sox2 Otx2
Sox site Otx site
Rax
Otx2
Sox site Otx site
Rax
Fig. S6. A model for the mutual inhibition between the Sox2 protein and Sox-binding site. Luciferase assays demonstrate that whereas transcription fromSOP-FLASH is induced by a combination of Otx2 and Sox2 but not by Otx2 alone, transcription from mS-FLASH is induced by Otx2 alone and not by a combinationof Otx2 and Sox2. The absence of either the Sox2 protein or Sox-binding site results in loss of transcription. However, in the absence of both these factors, Otx2alone can activate transcription.
Danno et al. www.pnas.org/cgi/content/short/0710954105 9 of 10
Input IP (myc)
HA-Sox2+-
++ myc-Otx2
--
+-
++
--
IP (myc)
HA-Sox2
myc-Otx2+-
++
--
IB: HA
IB: myc
Fig. 4C
myc-Otx2+-
++ HA-Sox2
--
+-
++
--
Input IP (HA)
IB: myc
myc-Otx2+-
++ HA-Sox2
--
IP (HA)
IB: HA
Fig. 4DFig. 5D
pulldownInput
Sox2 TW
P47R
P79L
TW
P47R
P79L
IB: HA
IB: Sox2
IB: α-tubulin
+2xt
O
HEK293T
)S
E esuom( l ortn oc
Fig. S3B
+2x o
S
HEK293T
( lortnoc2xt
O-cym
)
IB: Otx2
IB: α-tubulin
Fig. S3A
TS
G
GM
H-2xoS-
TS
G
-G
MH-2xo
S-T
SG
α∆1
-G
MH-2xo
S-T
SG
∆liat liat-
GM
H-2xoS-
TS
G
IB: myc
Coomassie
+ myc-Otx2
Fig. S5F
+ HA-Sox2
tupnI
TS
G
TW-2xt
O-T
SG
1-D
H-2xtO-
TS
G
2-D
H-2x tO-
TS
G
3 -D
H-2 xtO-
TS
G
4 -D
H-2 xtO-
TS
G
Coomassie
IB: HA
Fig. S5I
tupnI
TSG
2xtO-TSG- + - + HA-Sox2
IB: HA
Fig. 4A
tupnI
TSG
2xoS-TSG- + - + myc-Otx2
IB: myc
Fig. 4B
IB: HA
Coomassie
TS
G
TW-2xt
O-TS
G
N-2xtO-T
SG
1-D
H-2xtO-T
SG
4-D
H-2xtO-T
SG
1M-2xt
O-TS
G
2M-2xt
O-TS
G
C-2xtO-T
SG
tupnI
+ HA-Sox2
Fig. S5D
tupnI
TS
G
TW-2xo
S-T
SG
N-2xoS-
TS
G
GM
H-2xoS-
TS
G
C-2xoS-
TS
G
IB: Flag
Coomassie
+ Flag-Otx2
Fig. S5B
75
50
37
25
75
50
37
25
75
50
37
25
20
75
50
37
25
20
75
5037
2520
75
5037
2520
75
50
37
2520
75
50
37
2520
4530
45
30
4530
45
30
20
75
50
37
2520
75
50
37
2520
50
37
2520
50
37
2520
45
30
20
453020
45
30
Fig. S7. Full scans of the Western blotting data represented in Fig. 4 A–D, Fig. 5D, Fig. S3 A and B, and Fig. S5 B, D, F, and I.
Danno et al. www.pnas.org/cgi/content/short/0710954105 10 of 10
