The mechanisms of transcriptional regulation by the nuclear orphan receptor ROR(alpha)

A mutation in the constitutively active nuclear orphan receptor RORα results in a severe impairment of cerebellar development by unknown mechanisms. Mutational analysis carried out in this work unmasked the protein\u27s ability to repress in a cell type specific manner. Further studies demonstrated RORα\u27s ability to interact with nuclear corepressors N-CoR and SMRT. Similar studies were performed with the highly related nuclear orphan receptor RevErb, demonstrating that N-CoR is a RevErb corepressor. A strong activation domain in RORα\u27s C-terminus was also identified. We next searched for mammalian RORα coactivators using the minimal activation domain as bait in a yeast two-hybrid screen. Several known and previously unidentified putative coactivators were isolated, including GRIP-1 and the then novel protein subsequently named PBP. PBP was cloned and its interaction with thyroid hormone receptor was characterized. PBP and GRIP-1 interactions with RORα were confirmed in vitro and required the intact activation domain of RORα. Even in the absence of exogenous ligand, RORα interacted with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we identified several potential RORα coactivators that, in contrast to the interactions with hormone receptors, interact with RORα in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for RORα both in yeast and in mammalian cells. Thus, GRIP-1 was demonstrated to be the first proven RORα coactivator. Finally we attempted to clone a novel RORα interacting protein and discuss its potential role as a RORα coactivator. The results of this work contribute to our understanding of the overall mechanisms of transcriptional regulation by nuclear hormone receptors, specifically the nuclear orphan receptor RORα