Conservation and cis-regulatory activity of the sequences upstream of Rax. (A) Comparison of three upstream sequences of frog Rax (X. laevis, AY250711 and RaxG4; X. tropicalis, XtRaxG). A proximal region that contains a minimal promoter and transcriptional start site (red), as well as a distal noncoding region (blue), are highly conserved among the three clones. The distal region is termed the conserved noncoding sequence 1 (CNS1). The percentages indicate sequence similarities. (B) Transgenesis in X. laevis. Two constructs contain CNS1 and up-regulate the EGFP reporter. The number of transgenic embryos that express EGFP in the optic vesicles of normally developing embryos obtained in this assay is indicated on the right. Black lines indicate the Rax promoter. (C) Luciferase reporter activities of sequences upstream of Rax in X. laevis embryos. (D) VISTA view of the occurrence of the conserved sequence domain in the genomic region that encompasses the Rax gene. Colored peaks (purple, coding; pink, noncoding) indicate regions of at least 100 bp and 60% similarity. There are three conserved noncoding regions (arrowheads) in a region of ≈20 kb, and the most proximal one is CNS1.

Synergistic actions of Otx2 and Sox2 on transcription via CNS1. (A) Diagram of luciferase reporter constructs used in the present work. (B–D) Luciferase assays using Xenopus animal cap (AC) cells. Transcription from SOP-FLASH (B) is induced by the injection of Otx2 mRNA (100 pg) but not by the injection of Sox2 mRNA (100 pg). Overexpression of Otx2 does not induce transcription from either mO-FLASH (C) or mS-FLASH (D). (E) RT-PCR analysis shows that dominant-negative Sox2 (dn-Sox2) represses Otx2-induced Rax expression in animal cap cells. The amounts of mRNA injected were: Otx2, 100 pg; dn-Sox2, 1,000 pg; and Sox2, 10 or 30 pg. (F–H) Luciferase assays using HEK293T cells. (F) Synergistic effect of Otx2 and Sox2 on transcription from SOP-FLASH. (G) Transcription from mO-FLASH is not induced by the combination of Otx2 and Sox2 in HEK293T cells. (H) From mS-FLASH, transcription is induced by transfection with Otx2 alone in HEK293T cells. This increase is attenuated rather than enhanced by the addition of Sox2.

Missense mutations in the Sox2 HMG domain affect the activities of Sox2 protein associated with Otx2. (A) Missense mutations identified in helices 2 and 3 of the human Sox2 HMG domain (R74P and L97P). (B) The 3D structure of the Sox2 HMG domain (yellow) binding to DNA (gray), based on a previous study (30). The side chains of the 74th arginine (blue) and 97th lysine (red) do not come in direct contact with the DNA. (C) Mutated Sox2 proteins do not induce transcription via CNS1 in cooperation with Otx2. (D) GST pulldown assay showing that the mutated Sox2 proteins lose Otx2-binding activity. HA-tagged Sox2 proteins were in vitro-synthesized and pulled down by GST-Otx2, followed by Western blotting with antibody against HA-tag. (E) Quantification of Otx2-binding levels by using the Odyssey infrared imaging system. Error bars indicate SD values. (F) Molecular relationships among ocular malformation-associated genes. The present work demonstrates that a direct interaction between the Otx2 and Sox2 proteins coordinately regulates Rax expression (colored). Upstream proteins and target genes are indicated as round rectangles and ovals, respectively. A full scan of the Western blotting data is shown in Fig. S7.

Otx2 and Sox2 are upstream regulators of Rax. (A) Multiple DNA sequence alignment of vertebrate CNS1. CNS1 contains a specially conserved 35-nucleotide sequence (pentatriacontamer, pt) that contains consensus binding sites for Otx and Sox. (B) Genomic structure of the Xenopus Rax locus. Arrows indicate the primers used in the ChIP assays. CNS1, the coding region and untranslated region of Rax are indicated as black, gray, and white boxes, respectively. (C) The ChIP assay demonstrates that endogenous Otx2 and Sox2 proteins bind to CNS1 in vivo. (D) RT-PCR analysis showing that overexpression of Otx2, but not of Sox2, induces Rax in Xenopus animal cap cells. (E) Luciferase assays using Xenopus animal cap (AC) cells shows that overexpression of Otx2 induces transcriptional activation of pRax-2600b-Luc, whereas Sox2 overexpression does not. (D and E) Sox2 or Otx2 mRNA (100 pg) was injected.

Physical interactions between Otx2 and Sox2 proteins in vitro and in vivo. (A) GST pulldown by immobilized GST-Otx2 of in vitro-translated HA-Sox2 protein. (B) GST pulldown by immobilized GST-Sox2 of in vitro-translated myc-Otx2 protein. (C and D) Coimmunoprecipitation assays demonstrating in vivo interactions between the Otx2 and Sox2 proteins. (C) HA-Sox2 is coimmunoprecipitated with myc-Otx2 by the anti-myc antibody (9E10). (D) Myc-Otx2 is coimmunoprecipitated with HA-Sox2 by the anti-HA antibody (Y-11). Full scans of the Western blotting data are presented in Fig. S7.