FIG. 6.
KLF9 interacts functionally and physically with the basic domain adjacent to the Cf of GATA4. (A) Schematic diagram of various GATA deletion mutants and the ability to stimulate the mouse Dio1 promoter in association with KLF9 and HNF4α as evaluated by luciferase reporter assay. TAD, transcriptional activation domain. The data shown are the mean of duplicate experiments. Error bars indicate the range of the duplicates. (B) Interaction of GATA4 and KLF9 in transfected HEK293 cells. HEK293 cells were transfected with expression plasmids for GATA4 along with FLAG-tagged KLF9. Cells were harvested, and whole-cell lysate were immunoprecipitated with polyclonal anti-GATA4 (AF2606; R&D Systems) or control IgG (top) or monoclonal anti-FLAG M2 or control IgG (bottom) as described in Materials and Methods. The pellets from immunoprecipitation were subjected to SDS-PAGE and immunoblotted with anti-FLAG (top) or anti-GATA antibody (bottom). The expression of GATA4 or KLF9 was confirmed by immunoblot analysis of 10 μg of whole-cell lysate (Input) with anti-FLAG or anti-GATA4 antibody (AF2606), respectively. Ab, antibody; IP, immunoprecipitation; Cont, control. (C and D) Mapping of the region of GATA4 required for the interaction with KLF9 by coimmunoprecipitation analyses. HEK293 cells expressing FLAG-KLF9 and various GATA4 deletion mutants were immunoprecipitated with polyclonal antibody directed against the C-terminal part of GATA4 (aa 328 to 439) (sc-9053; Santa Cruz Biotechnology) (C) or the N-terminal part of GATA4 (aa 27 to 211) (AF2606) (D) as described for panel B. To detect KLF9 in the protein complex, peroxidase-conjugated anti-FLAG M2 antibody was used, and to detect GATA4, monoclonal anti-GATA4 (IgG-H2429) (for panel C) and polyclonal GATA4 (AF2606) antibodies (for panel D) were used for the immunoblot analyses. (E) Nuclear fractions were prepared from lysates of HEK293 cells expressing full-length (aa 1 to 442) or mutant (Δ304-332) GATA4. The presence of GATA4 in these subcellular fractions was detected by immunoblotting with an anti-GATA4 antibody. The nuclear fraction was confirmed by detection of the nuclear protein nucleoporin. (F) GST pull-down assays were performed with various 35S-labeled GATA4 mutants in the presence of GST or GST fusions containing either the full length (aa 1 to 244) or the deletion mutant form of KLF9 [KLF9(Δ1-41)] immobilized on glutathione beads. After washing, specifically bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography. The input samples contain 1% of the material added to each GST assay. ZF, zinc finger.