Biochemical Isolation and Analysis of a Nuclear Receptor Corepressor Complex

This chapter describes the initial isolation and characterization of a core Silencing Mediator of Retinoid and Thyroid Receptors (SMRT) corepressor complex from HeLa cells. The isolation of native SMRT results in the copurification of the WD40 repeat protein—TBL1 (Transducin Beta-Like protein-1) and histone deacetylase3 (HDAC3). Because many HDACs are either severely impaired or inactive when produced and purified alone, the chapter discusses a reconstitution of HDAC3 activity using a rabbit reticulocyte lysate coupled transcription–translation system. This system is used to show that SMRT/N-CoR activates HDAC3 activity without the aid of TBL1, and it can be readily adapted to screen for other potential activators of HDAC activity. The chapter also shows the ways to produce a large-scale preparation of an active SMRT–HDAC3 complex using the baculovirus infection of sf9 cells. A fundamental action of many nuclear hormone receptors is the establishment of active repression in the absence of ligand. While nuclear receptors themselves can exhibit repressive behavior via contacts with the basal transcription machinery, the corepressors N-CoR (Nuclear Receptor CoRepressor) and SMRT have emerged as the primary mediators of nuclear receptor imposed repression. Many HDACs are either inactive or have low enzymatic activity when produced recombinantly.