Identification of Reverbα as a novel RORα target gene

The nuclear receptor superfamily comprises a large number of ligand-activated transcription factors that are involved in numerous biological processes such as cell proliferation, differentiation, and homeostasis. RORalpha (NR1F1) and Reverbalpha (NR1D1) are two members of this family whose biological functions are largely unknown. In addition, no ligand has been yet identified for these two receptors; therefore, they are referred as orphan receptors. Here, we show that RORalpha and Reverbalpha are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells. Ectopic expression of RORalpha1 in L6 cells significantly induces Reverbalpha expression as demonstrated by Northern blot analysis. Using reverse transcription-PCR to analyze Reverbalpha gene expression from staggerer mice, we found that there was a significant reduction of Reverbalpha mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that RORalpha is involved in the regulation of Reverbalpha gene expression. Transient transfection assays using the Reverbalpha promoter demonstrate that RORalpha regulates the Reverbalpha gene at the transcriptional level. Furthermore, mutagenesis experiments indicate that RORalpha regulates Reverbalpha transcription via a monomeric ROR response element located in the Reverbalpha gene promoter. Electrophoretic mobility shift assays show that RORalpha binds strongly to this site in a specific-manner. Finally, overexpression of GRIP-1/TIF-2, but not SRC-1, potentiates RORalpha-stimulated Reverbalpha promoter activity in transient transfection experiments. Together, our results identify Reverbalpha as a novel target gene for RORalpha.