FIGURE 6.

FBI-1 interacts with the co-repressors via its POZ-domain, and BCoR is recruited to the promoter by FBI-1. A, diagram of the mammalian two-hybrid assay and protein-protein interaction between the POZ-domain of FBI-1 and the co-repressors. Upstream activating sequence (UAS), binding site for Gal4DBD (B) GST fusion protein pulldown assays and structure of the co-repressors SMRT, NCoR, BCoR, and FBI-1. GST and GST-POZFBI-1 fusion proteins were incubated with in vitro synthesized [³⁵S]methionine-labeled co-repressor polypeptides and pulled down. RD, repression domain; N3-1 and S1–2, domains involved in interaction with nuclear receptors; ZF, zinc finger domain; POZ, POZ-domain of FBI-1. Domains of co-repressors used for in vitro pulldown assays are indicated below by light gray filled bars. C, co-immunoprecipitation of FBI-1 and BCoR. Cell lysates prepared from HEK293A cells transfected with FLAG-FBI-1 and Myc-BCoR expression vectors were immunoprecipitated using anti-Myc antibody and analyzed by Western blotting using anti-FLAG antibody. D, ChIP assays of BCoR and FBI-1 binding at the distal FRE elements and 3′-UTR of Rb gene. HEK293A cells were transfected with FLAG-FBI-1 and/or Myc-BCoR expression vector and immunoprecipitated with the antibodies indicated. More BCoR is bound to the FRE cluster in the presence of FBI-1. A histogram of the ChIP assays is shown on the right.