Supplemental Methods
Details of the functional studies
Primers and probes
The primers and probes utilized in this study are summarized in Supplemental Table 1.
Vectors
The expression vector and the fluorescent vector containing the wild-type (WT) OTX2 cDNA wereconstructed by fusing the OTX2 cDNA to the Myc tag in pCMV-Myc and to the green fluorescentprotein (GFP) gene in pAcGFP1-C1 (Clontech, Palo Alto, CA), respectively (designated aspOTX2-WT and pGFP-WT, respectively). The wild-type OTX2 cDNA was obtained from a humanpituitary cDNA sample (Clontech), using primers that were designed to lose the first codon to enablethe fusion to the C-terminal sides of the Myc tag and the GFP gene. The expression vector and thefluorescent vector containing the mutant OTX2 cDNA (designated as pOTX2-R90Sand pGFP-R90S,respectively) were created by site-directed mutagenesis, using the Prime STAR Mutagenesis Basal kit(Takara, Otsu, Japan). The luciferase reporter vectors were constructed by inserting the promotersequences of IRBP (–138 to +82 bp), HESX1 (–405 to +267 bp), and POU1F1 (–541 to +6 bp) into pGL3 basicthat includes cDNA encoding firefly luciferase as a reporter gene(Promega, Madison, WI)(designated as pIRBP-luc, pHESX1-luc, and, pPOU1F1-luc,respectively).
Western blot analysis
The Myc-tagged pOTX2-WT and pOTX2-R90S(20 µg) were transfected into COS1 cells in a 10cm plate. Cell lysates were obtained at 48 hours after transfection, and probed withantibodies for Myc and β-actin utilized as an internal control.
Subcellular localization analysis
The pGFP-WT and pGFP-MT (4 µg) were transfected into COS1 cells in a 3.5cmdish. The fluorescent signals were observed at 48–72 hours after the transfection using a laser-scanningmicroscope LSM510 (Version 3.2) (Carl Zeiss, Oberkochen, Germany) shortly after nuclear stainingwith 4, 6 diamidino-2-phenylindole(DAPI).
DNA binding analysis
Electrophoretic mobility shift assay (EMSA) was performed, using probes within the IRBP, HESX1,and POU1F1 promoter sequences (designated as IP1-WT, HX1-WT, and PF-1-WT, respectively), and nuclear extracts which were obtained from COS1 cells transfected with pOTX2-WT or pOTX2-R90S, using Nuclear and Cytoplasmic Extraction Reagents(Pierce, Rockford, IL). For comparison, EMSA was also performed with mutant probes in which theOTX2 binding sites were destroyed (designated as IP1-MT, HX1-MT, and PF1-MT). Eachbiotin-labeled probe (20 fmol) was incubated with 2 μlof nuclear extract with wild-type ormutant OTX2 protein, and the incubation mixture was subjected to polyacrylamidegel electrophoresis. After electrophoretic transfer to a nylon membrane, thebiotin-labeled probe was detected using the Streptavidin-Horseradish Peroxidase Conjugate and the Chemiluminescent Substrate of Lightshiftchemiluminescent EMSA kit (Pierce).
Transactivation analysis
Transactivation analysis was performed with Dual-Luciferase Reporter Assay System (Promega). COS1 cells seeded in 12-well dishes (1.5x105 cells/well) were transiently transfectedusing lipofectamine 2000 (Invitrogen) with [1] the empty expression vector (0.6 µg), [2] pOTX2-WT(0.6 µg), [3] pOTX2-R90S(0.6 µg), or [4] pOTX2-WT (0.3 µg) plus pOTX2-R90S(0.3 µg), togetherwith each reporter vector (0.6 µg) (pIRBP-luc, pHESX1-luc, or pPOU1F1-luc), andan internal control vector for the transfection (pRL-CMV)(20 ng), which includes cDNA encoding renilla luciferase(Promega, Madison, WI). Luciferase assays wereperformed at 48 hours after the transfection with Lumat LB9507 (Berthold, Bad Wildbad, Germany).Transfections were performed in triplicate within a single experiment, and the experiment wasrepeated three times.