DISTINCT STEADY-STATE NUCLEAR RECEPTOR COREGULATOR COMPLEXES EXIST IN-VIVO

Transcriptional regulation by members of the nuclear hormone receptor superfamily is a modular process requiring the mediation of distinct s ubclasses of coregulators, These subclasses include members of the ste roid receptor coactivator-1 (SRC-1) coactivator family, p300/CBP and t heir associated proteins, such as p300/CBP-associated factor, human ho mologs of SWI/SNF proteins such as BRG-1, and the less well-characteri zed E3 ubiquitin-protein ligases E6 papillomavirus protein-associated protein and receptor-potentiating factor-1. Because functional studies indicate that these coregulators may form higher order complexes, we analyzed steady-state complexes of different coregulator subclasses in vivo. T47D and HeLa cell lysates were subjected to biochemical fracti onation and screened by immunoblotting using coregulator-specific anti bodies. We show that different subclasses of nuclear receptor coregula tors exhibit distinct fractionation profiles. Furthermore, evidence is provided that SRC-1 family members may exist in vivo in heteromultime ric forms with each other. In addition, we demonstrate that liganded P R is present in stable complexes containing SRC-1 and transcription in termediary factor 2 (TIF2) ill vivo. Our results suggest that the asse mbly of large, modular transcriptional complexes by recruitment of dis tinct subclasses of preformed coregulator subcomplexes may be involved in transcriptional regulation by activated nuclear receptors.