TRANSCRIPTIONAL REPRESSION BY REV-ERBA-ALPHA IS DEPENDENT ON THE SIGNATURE MOTIF AND HELIX-5 IN THE LIGAND-BINDING DOMAIN - SILENCING DOES NOT INVOLVE AN INTERACTION WITH N-COR

Rev-erbA alpha is an orphan nuclear receptor that functions as a domin ant transcriptional repressor. Tissue culture and in situ hybridisatio n studies indicated that Rev-erbA alpha plays an important role in mam malian differentiation and development. Previous studies have localise d the silencing domain of Rev-erbA alpha to the D/E region of the orph an receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains t he ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxx LL] and a region that, according to the recently published crystal str uctures of steroid receptors, would be predicted to form helix 5 of th e canonical LBD structure. Fine deletions and site-specific mutagenesi s indicated that both the LBD signature motif and helix 5 were necessa ry for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepre ssor, N-CoR, that is known to interact with the thyroid hormone and re tinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. I n conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this or phan receptor.