TRANSCRIPTIONAL REPRESSION BY THE ORPHAN STEROID-RECEPTOR RVR REV-ERB-BETA IS DEPENDENT ON THE SIGNATURE MOTIF AND HELIX-5 IN THE E-REGION - FUNCTIONAL EVIDENCE FOR A BIOLOGICAL ROLE OF RVR IN MYOGENESIS/

RVR/Rev-erb beta/BD73 is an orphan steroid receptor that has no known ligand in the 'classical' sense. RVR binds as a monomer to an element which consists of an A/T-rich sequence upstream of the consensus hexam eric half-site. However, RVR does not activate transcription and block s transactivation of this element by ROR/RZR. The mechanism of RVR act ion remains obscure, hence we used the GAL4 hybrid system to identify and characterize an active transcriptional silencer in the ligand bind ing domain (LED) of RVR. Rigorous deletion and mutational analysis dem onstrated that this repressor domain is encoded by amino acids 416-449 of RVR. Furthermore, we demonstrated that efficient repression is dep endent on the so-called LED-specific signature motif, (F/W)AKxxxxFxxLx xxDQxxLL (which spans loop3-4 and helix 4) and helix 5 (H5; identified in the crystal structures of the steroid receptor LBDs). Although RVR is expressed in many adult tissues, including skeletal muscle, and du ring embryogenesis, its physiological function in differentiation and mammalian development remains unknown. Since other 'orphans', e.g. COU P-TF II and Rev-erbA alpha, have been demonstrated to regulate muscle and adipocyte differentiation, we investigated the expression and func tional role of RVR during mouse myogenesis. In C2C12 myogenic cells, R VR mRNA was detected in proliferating myoblasts and was suppressed whe n the cells were induced to differentiate into post-mitotic, multinucl eated myotubes by serum withdrawal. This decrease in RVR mRNA correlat ed with the appearance of muscle-specific markers (e.g. myogenin mRNA) . RVR 'loss of function' studies by constitutive over-expression of a dominant negative RVR Delta E resulted in increased levels of p21(Cip1 /Waf1) and myogenin mRNAs after serum withdrawal. Time course studies indicated that expression of RVR Delta E mRNA results in the precociou s induction and accumulation of myogenin and p21 mRNAs after serum wit hdrawal. In addition, we demonstrated that over-expression of the COUP -TF II and Rev-erbA alpha receptors in C2C12 cells completely blocked induction of p21 mRNA after serum withdrawal. In conclusion, our studi es identified a potent transcriptional repression domain in RVR, chara cterized critical amino acids within the silencing region and provide evidence for the physiological role of RVR during myogenesis.