Requirement of activation domains of SOX2 and Pax6 for synergistic activation of the DC5 enhancer. (A) Schemes of the deletion mutants of SOX2 and Pax6, which have the 3xHA tag at their C-termini. (B) Various combinations of effector vectors to express SOX2 and Pax6 in wild-type or mutant forms were transfected into chicken liver cells with the DC5 enhancer-containing luciferase reporter plasmid. (C) Effector vectors for the wild-type and mutant forms of Pax6 were transfected into chicken liver cells with the Pax6(PD)CON sequence-containing luciferase reporter plasmid.

Formation of the high-mobility ternary complex in vitro that correlates with synergistic activation of the DC5 enhancer by SOX2 and Pax6 in cultured cells. (A,B) Differences in ternary complex formation on wild type and mutant sequences of DC5. (A) Nucleotide replacement mutants (M4 and M7); (B) nucleotide insertion mutants (I2, I4, and I10) (Fig. 1). High-mobility and low-mobility forms of the ternary complex are indicated by superscripts H and L, respectively. (C) Wild type and mutant DC5 enhancers in (A) and (B) were tested for activation by exogenous SOX2 and Pax6 in liver cells. (D) Alteration of the Pax6 binding sequence of DC5 closer to the consensus binding sequence [DC5-Pax6(NC)] increases the binding of Pax6(169) itself, but disrupts cooperative binding of SOX2(202) and Pax6(169). (E) Loss of the cooperative binding on the DC5-Pax6(NC) correlates with loss of synergistic activation by SOX2 over activation by Pax6 itself.

Induction of ectopic δ-crystallin expression by exogenous SOX2 and Pax6 in chicken embryo ectoderm. (A–F) Chicken embryos were electroporated in ovo at stage 10 with vectors expressing SOX2 and Pax6 (A,B), SOX2 alone (C,D), or Pax6 alone (E,F), and then analyzed at stage 17 for expression of the δ-crystallin gene by whole mount in situ hybridization (A,C,E). Ectopic δ-crystallin expression is indicated by yellow arrows. Electroporated cells were marked by GFP expression (B,D,F). (G,H) Sections of the embryo expressing ectopic δ-crystallin shown in (A); section showing endogenous δ-crystallin expression in the lens (G), and section showing ectopic δ-crystallin expression in the ectodermal cells with the morphology of thickened placode (H). (I) Low level expression of Pax6 in the head ectoderm of stage 17⁻ normal embryo (green arrow). (J,K) Up-regulation of Pax6 expression in the head ectoderm by exogenous SOX2 in a stage 17⁺ embryo (J, green arrow). The low level Pax6 expression observed in (I) is extinct by this stage. Electroporated cells were marked by GFP expression (K).

Interdependent association of SOX2 and Pax6 with the DC5 enhancer in a chromatin environment in vivo. (A) The DC5-lacZ reporter construct introduced into yeast genome and the location of the sequences amplified by PCR. (B,C) Chromatin immunoprecipitation analysis using yeast cells carrying various combinations of SOX2, Pax6, and empty expression vectors as indicated: –, empty vectors; SOX2-HA and SOX2-FLAG, C-terminally 3xHA- and 3xFLAG-tagged SOX2 expression vectors, respectively, containing the TRP1 selection marker; GAL4AD-Pax6-HA and GAL4AD-Pax6-FLAG, C-terminally 3xHA- and 3xFLAG-tagged GAL4AD-Pax6 expression vectors, respectively, containing the LEU2 selection marker. Immunoprecipitation was performed with anti-HA antibody and PCR was done on the DC5 sequence (B) and the lacZ gene (C). PCR products from 2.5-fold serial dilutions of total chromatin demonstrate the linear response of the PCR. (D) Expression of the reporter lacZ gene in yeast cells having the combinations of SOX2/Pax6 effectors and used for chromatin immunoprecipitation.

Cooperative binding of SOX2 and Pax6 to the DC5 sequence in vitro. (A) SOX2 and Pax6 proteins and their variants with oligopeptide tags. DNA binding domains are shaded: PD, paired domain; HD, homeodomain; HMG, HMG domain. (B) Difference of affinity of the Pax6 paired domain in binding to the consensus sequence [Pax6(PD)CON] and to the DC5 sequence indicated by gel mobility shift assays. (C) Cooperative binding of the Pax6 paired domain and the SOX2 HMG domain to the DC5 sequence in vitro. (D) FLAG-tagged Pax6(169) and His-tagged SOX2(202) were used to make a complex on DC5 probe, and effect of anti-tag antibodies on the complex was examined. (E) Direct interaction of the Pax6 paired domain and the SOX2 HMG domain without intervention of DNA. MBP-tagged proteins were incubated with GST-SOX2 or GST and precipitated with glutathione sepharose. Effect of Escherichia coli DNA in the protein preparations was quenched by EtBr.

A model of a genetic switch composed of SOX2/SOX3 and Pax6, which regulates initiation of lens development. (A) Expression of Pax6 (green), Sox2 and Sox3 (red), and δ-crystallin (blue) in the ectoderm during lens induction in chicken embryos. A very low level of Sox2 expression observed in the stage 11 head ectoderm is not illustrated. Expression of the genes in the optic vesicle is omitted. (B) Molecular events that occur on the δ-crystallin enhancer during early lens development. At stage 11, Pax6 protein is expressed in the head ectoderm, but does not make a stable complex with the target DNA sequences such as DC5. After Sox2/Sox3 expression commences at stage 12, Pax6 cooperatively binds with SOX2/SOX3 to the target DNA sequences, and activates genes for lens differentiation such as δ-crystallin. Ternary complex formation presumably involves conformational changes of Pax6 and SOX2/SOX3 as well as DNA.

Identification of Pax6 as SOX1/SOX2/SOX3-partner factor δEF3 by functional expression screen. (A) Functional expression screen using yeast cells. (a) The scheme of the screening procedure. Yeast cells carrying the DC5 enhancer-dependent HIS3 and lacZ reporters were first transformed with a SOX1 or SOX2 expression vector. GAL4 activation domain-fused lens cDNA library was then introduced. Yeast cells that harbor both SOX1/SOX2 vector and a δEF3 clone from the library are expected to express HIS3 and lacZ genes. (b) Wild-type (WT) and mutant sequences of the DC5 enhancer used in this study. Altered or inserted nucleotides are shaded. SOX and δEF3 (Pax6) binding regions are shown at the top. (c–e) The growth and β-galactosidase expression of the HIS3/lacZ reporter-containing yeast cells, which harbor expression plasmids for various Sox and Pax6 cDNAs. (c,d) The growth of three clones in the presence (c) and in the absence (d) of histidine; (e) expression of β-galactosidase. (B) Cell type-independent activation of the DC5 enhancer by SOX2 and Pax6. (a) Structures of the luciferase reporter and effector plasmids. (b,c) Varying amounts of effector plasmids to express SOX2 or Pax6 and the reporter plasmids were transfected into lens cells (b) and liver cells (c). The luciferase activity generated by the enhancer-less reporter was taken as 1. (C) Binding of Pax6 to the DC5 sequence and sequence-specific competition. The probe was DC5-WT sequence. 50 ng of poly dA-dT was included in the reaction to achieve a stringent binding condition.