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First published online 22 February 2005
doi: 10.1242/jcs.01706

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PITX2, ß-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter

¹ Department of Biological Science, The University of Tulsa, 600 S College Ave., Tulsa, OK 74104-3189, USA
² Department of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
³ Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, IA 52242, USA

View larger version (15K): [in a new window]   Fig. 1. Activation of the LEF-1 promoter by PITX2. (A) Human LEF-1 promoter elements used in the transfection assays. PITX2 binding sites (Bicoid sites) are denoted by an asterisk (*). (B) CHO cells were transfected with 5 µg of the appropriate human LEF-1 luciferase reporter constructs. The cells were co-transfected with 2.5 µg of either the CMV-PITX2A, or the CMV plasmid without PITX2 (vector control). CHO cell lysates transfected with empty vector were used as a control to show lack of endogenous PITX2 protein in CHO cells. To control for transfection efficiency, all transfections included the SV-40 ß-galactosidase reporter. Cells were incubated for 24 hours, and then assayed for luciferase and ß-galactosidase activities. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmids without PITX2 expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments). PITX2 expression did not change the levels of ß-galactosidase activity in the transfected cells.

View larger version (12K): [in a new window]   Fig. 2. PITX2 isoforms differentially regulate the LEF-1 promoter. CHO cells were transfected as described in Fig. 1, using the three major PITX2 isoforms. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmid without PITX2 expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments).

View larger version (10K): [in a new window]   Fig. 3. LEF-1 and PITX2 synergistically activate the LEF-1 promoter. CHO cells were transfected as in Fig. 1, with CMV-PITX2A or CMV-LEF-1, or both, and the CMV empty expression vector. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmid without PITX2 expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments). LEF-1 expression did not change the levels of ß-galactosidase activity in the transfected cells.

View larger version (44K): [in a new window]   Fig. 4. LEF-1 physically interacts with PITX2. (A) GST-LEF-1 pull-down assay with bacterial expressed and purified PITX2A protein (200 ng). PITX2A binds to immobilized GST-LEF-1, showing that PITX2A can physically interact with LEF-1. The bound protein was detected by western blot using the PITX2 antibody, P2R10. As a control GST-beads were incubated with purified PITX2A to show the specificity of binding to LEF-1. (B) GST-ß-catenin and GST-PITX2A pull-down assay with bacterial expressed and purified LEF-1 protein (50 ng). LEF-1 binds to immobilized GST-ß-catenin as expected and used as a positive control. LEF-1 binds to GST-PITX2A in a reciprocal experiment shown in A. The bound protein was detected by western blot using a LEF-1 antibody. As a control, GST-beads were incubated with purified LEF-1 to show the specificity of binding to ß-catenin and PITX2A.

View larger version (33K): [in a new window]   Fig. 5. LEF-1 binds to the C-terminal tail of PITX2. (A) The PITX2 deletion constructs used to map the LEF-1 interaction. (B) GST-PITX2A, GST-PITX2 HD (homeodomain only), GST-PITX2 C173 (C-terminal tail only) and GST-PITX2 173 (deletion of the C-terminal tail) pull-down assay with bacterial expressed and purified LEF-1 protein (50 ng). LEF-1 binds to GST-PITX2A and GST-PITX2 C173 but not to GST-PITX2 HD or GST-PITX2 173. The bound protein was detected by western blot using a LEF-1 antibody. As a control GST-beads were incubated with purified LEF-1 to show the specificity of binding to PITX2A.

View larger version (15K): [in a new window]   Fig. 6. The LEF-1 WRE represses PITX2 activation. (A) The WRE deletion in the LEF-1 promoter compared with the full-length LEF-1 promoter. (B) CHO cells were transfected as in Fig. 1, with CMV-PITX2 isoforms and the CMV empty expression vector with the appropriate LEF-1 luciferase reporter constructs. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmid without PITX2 expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments).

View larger version (26K): [in a new window]   Fig. 7. ß-catenin and PITX2 synergistically activate the LEF-1 promoter independent of the WRE. (A) CHO cells were transfected as in Fig. 6, with CMV-PITX2C or CMV-ß-catenin S37A, or both, and the CMV empty expression vector with the appropriate LEF-1 luciferase reporter constructs. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmid without PITX2 or ß-catenin expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments). (B) GST-ß-catenin pull-down assay with bacterial expressed and purified PITX2A protein (200 ng). PITX2A binds to immobilized GST-ß-catenin. The bound protein was detected by western blot using a PITX2 antibody. As a control GST-beads were incubated with purified PITX2A to show the specificity of binding to ß-catenin.

View larger version (11K): [in a new window]   Fig. 8. Combinatorial effect of PITX2, LEF-1 and ß-catenin on LEF-1 promoter activity. (A) CHO cells were transfected as in Fig. 1, with CMV-PITX2C, CMV-LEF-1 or CMV-ß-catenin S37A, or combinations of each, and the CMV empty expression vector. The activities are shown as mean fold activation compared with the LEF-1 promoter plasmid without protein expression and normalized to ß-galactosidase activity (± s.e.m. from four independent experiments). (B) Expression of PITX2 in transfected CHO cell lysates; approximately 10 µg of lysate was used in the western blot. PITX2 expression was similar in cells transfected with LEF-1 or ß-catenin, or both. Bacteria expressed PITX2 protein (100 ng) was used as a control; the protein expressed in transfected cells migrates slower than the bacterially purified protein due to the presence of a Myc/His C-terminal tag.