Identification of Reverbα as a Novel RORα Target Gene

Philippe Delerive,William W Chin,Chen S Suen

doi:10.1074/jbc.m202979200

Abstract

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. ROR(alpha) (NR1F1) and Reverb(alpha) (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 ROR(alpha) and Reverb(alpha) are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells. Ectopic expression of ROR(alpha)1 in L6 cells significantly induces Reverb(alpha) expression as demonstrated by Northern blot analysis. Using reverse transcription-PCR to analyze Reverb(alpha) gene expression from staggerer mice, we found that there was a significant reduction of Reverb(alpha) mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that ROR(alpha) is involved in the regulation of Reverb(alpha) gene expression. Transient transfection assays using the Reverb(alpha) promoter demonstrate that ROR(alpha) regulates the Reverb(alpha) gene at the transcriptional level. Furthermore, mutagenesis experiments indicate that ROR(alpha) regulates Reverb(alpha) transcription via a monomeric ROR response element located in the Reverb(alpha) gene promoter. Electrophoretic mobility shift assays show that ROR(alpha) binds strongly to this site in a specific-manner. Finally, overexpression of GRIP-1/TIF-2, but not SRC-1, potentiates ROR(alpha)-stimulated Reverb(alpha) promoter activity in transient transfection experiments. Together, our results identify Reverb(alpha) as a novel target gene for ROR(alpha).

Highlights

Members of the nuclear receptor (NR)1 superfamily have been shown to be involved in a wide range of biological processes such as cell proliferation, differentiation, and homeostasis [1, 2]
We show that ROR␣ and Reverb␣ are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells
Using reverse transcription-PCR to analyze Reverb␣ gene expression from staggerer mice, we found that there was a significant reduction of Reverb␣ mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that ROR␣ is involved in the regulation of Reverb␣ gene expression

Summary

Introduction

Members of the nuclear receptor (NR)1 superfamily have been shown to be involved in a wide range of biological processes such as cell proliferation, differentiation, and homeostasis [1, 2]. Because both ROR␣ and Reverb␣ have been reported to be highly expressed in the skeletal muscle [5], we investigated their relative gene expression during myogenesis using a rat L6 myoblastic cell line. To provide genetic evidence for a potential role of ROR␣ in the control of Reverb␣ gene expression, Reverb␣ mRNA levels were analyzed in muscle from wild-type and homozygous staggerer mice by reverse transcription-PCR (Fig. 2D).

Results

Conclusion