FIG. 2.

The Runx2 carboxy terminus contains several portable repression domains. (A) Schematics of the heterologous GAL-Luc reporter and GAL-Runx2 (1-513) fusion proteins used in these experiments. The reporter contains a minimal herpes virus TK promoter between four GAL4 binding sites and the Luc reporter gene. Amino acid residue numbers are above the Runx2 diagram. The relative locations of the activation domain (AD), nuclear matrix targeting signal (NMTS), and the TLE binding repression domain (RD) are depicted. (B) The TLE binding domain is not required for GAL-Runx2-mediated repression of a heterologous reporter. NIH-3T3 cells were cotransfected with GAL-TK-Luc, pRL-TK, and 700 ng of the indicated pCMV5-M2 or pCMV5-M2-Runx2 expression constructs. Firefly luciferase activities were corrected for transfection efficiency with renilla luciferase values. The change in repression (n-fold) was calculated relative to that in cells transfected with pCMV5. (C) The relative expression levels of GAL-Runx2 fusion proteins were determined by immunoblot analysis with anti-GAL-DNA binding domain monoclonal antibody. (D) Runx2 contains multiple regions flanking the activation domain that contribute to repression. In these experiments, pCMV5-M2-Runx2 plasmids were added in various quantities (100, 300, or 700 ng). Values were corrected to SEAP activity, and the change in repression (n-fold) was calculated relative to that in cells transfected with pCMV5-GAL. (E) The relative expression levels of GAL-Runx2-C-terminal fusion proteins were determined by immunoblot analysis as described for panel C. (F) The nuclear matrix targeting signal is also a portable transcriptional repressor. These experiments were performed as described above for panel D.