2 RECEPTOR INTERACTION DOMAINS IN THE COMPRESSOR, N-COR RIP13, ARE REQUIRED FOR AN EFFICIENT INTERACTION WITH REV-ERBA-ALPHA AND RVR - PHYSICAL ASSOCIATION IS DEPENDENT ON THE E-REGION OF THE ORPHAN RECEPTORS/

Rev-erbA alpha and RVR/Rev-erb beta/BD73 are orphan steroid receptors that have no known ligands in the 'classical sense', These 'orphans' d o not activate transcription, but function as dominant transcriptional silencers, The thyroid hormone receptor (TR) and the retinoic acid re ceptor (RAR) act as transcriptional silencers by binding corepressors (e.g. N-CoR/RIP13 and SMRT/TRAC-2) in the absence of ligands, The mole cular basis of repression by orphan receptors, however, remains obscur e, and it is unclear whether these corepressors mediate transcriptiona l silencing by Rev-erbA alpha and RVR, Recently, two new variants of N -CoR have been described, RIP13a and RIP13 Delta 1. The characterisati on of these splice variants has identified a second receptor interacti on domain (ID-II), in addition to the previously characterised interac tion domain (ID-I), This investigation utilised the mammalian two hybr id system and transfection analysis to demonstrate that Rev-erbA alpha and RVR will not efficiently interact with either ID-I or ID-II separ ately from RIP13a or RIP13 Delta 1. However, they interact efficiently with a domain composed of ID-I and ID-II from RIP13a, Interestingly, the interaction of Rev-erbA alpha and RVR is strongest with ID-I and I D-II from RIP13 Delta 1. Detailed deletion analysis of the orphan rece ptor interaction with RIP13/N-CoR rigorously demonstrated that the phy sical association was critically dependent on an intact E region of Re v-erbA alpha and RVR, Over-expression of the corepressor interaction d omains (i.e. dominant negative forms of N-CoR/RIP13) could alleviate o rphan receptor-mediated repression of transactivation by GALVP16, This demonstrated that these regions could function as anti-repressors. In conclusion, these data from two independent approaches demonstrate th at repression by Rev-erbA alpha and RVR is mediated by an interaction of ID-I and ID-II of N-CoR, RIP13a and Delta 1 with the putative ligan d binding domain of the orphan receptors.