Fig. 7. BMP SMADs associate with the N terminus of YY1: (A) Co-immunoprecipitation of YY1 with the SMAD1/4 complex. Whole-cell extracts from either BMP-stimulated COS cells transfected with expression vehicles encoding either human YY1 (lanes 1-4), Myc-tagged (MT) SMAD1 (lanes 2 and 4) or myc/Flag-tagged (MT/F)-SMAD4 (lanes 3 and 4). SMAD-associated proteins were immunoprecipitated from cell extracts with an anti-myc monoclonal antibody, subjected to polyacrylamide gel eletrophoresis (PAGE) and detected following western blot with either anti-myc or anti-YY1 polyclonal antibodies. Levels of YY1 present in the input extract used for immunoprecipitation are shown in bottom panel. Bands corresponding to YY1, SMAD1 and SMAD4 are indicated by arrowheads on the right, as are molecular mobility markers. (B) Mapping the SMAD1/4 interaction domain of YY1 by co-immunoprecipitation. COS cells were transfected with expression vehicles encoding either wild-type (WT) Flag-tagged (F)-YY1(1-414) or various Flag-tagged deletion mutants of YY1, plus expression vehicles encoding MT/F-SMAD4 and MT-SMAD1, as indicated. SMAD-associated proteins were immunoprecipitated from cell extracts with an anti-Myc monoclonal antibody, subjected to polyacrylamide gel eletrophoresis (PAGE) and detected following western blot with anti-Flag monoclonal antibody (upper panel). Anti-Flag western blot of 2.5% of input used in co-immunoprecipitation confirms substantial expression of all tagged deletion mutants of YY1 and SMAD4 in the various cell extracts (lower panel); in addition, equivalent levels of MT-SMAD1 expression was detected in the various cell extracts (data not shown). Deletion mutant used in each sample is shown above the lanes; band representing Flag/MT SMAD 4 (Sm4) is shown by arrowheads on the right, as are those of YY1 isoforms, bracketed on the right. Results shown are representative of three independent co-immunoprecipitation experiments. (C) The SMAD1/4 interaction domain of YY1 maps between residues 1-170. Schematic representation of YY1 structural regions is shown at top [adapted, with permission, from Thomas and Seto (Thomas and Seto, 1999)]; solid bars showing extent of various YY deletion mutants assayed in B. Gray box identifies SMAD1/4-interacting region of YY1 based upon amino acids shared by all YY1 deletion mutants that associate with SMAD1/4. Relative strength of SMAD1/4 association of various YY1 deletion mutants is indicated on extreme left by either +++ (strong association), ++ (weak but detectable association), no mark (no detectable association). (D) Co-expression of YY1(261-414), which lacks the SMAD1/4 interaction domain, inhibits BMP induction of a CAR3-driven reporter. P19 cells were transfected with Nkx2.5-lux-CAR3, Tk-renilla-luciferase (for normalization) and expression vehicles encoding either SMADs1 and 4 and p21E1b (to inhibit apoptosis due to loss of YY1 activity), plus increasing amounts (0-100 ng/well) of expression vectors encoding either wild-type YY1(1-414) (right lanes) or N-terminally truncated YY1(261-414) (left lanes). YY1(261-414) is able to bind to Site D in SR3 (data not shown) but is incapable of SMAD1/4 interaction (see above), and blocks the ability of BMP signals to induce the expression of a CAR3-driven reporter.