Identification of a retinoic acid response domain involved in the activation of the beta 1-adrenergic receptor gene by retinoic acid in F9 teratocarcinoma cells.

The density of beta 1-adrenergic receptors (beta 1-AR) is up-regulated upon differentiation of embryonic F9 teratocarcinoma cells by retinoic acid (RA) to the primitive endodermal phenotype. To identify the domains involved in RA-mediated activation of beta 1-AR gene transcription, three kb of 5'-flanking sequence of the beta 1-AR gene were ligated to a luciferase reporter gene and transiently transfected into F9 cells that were pre-exposed to 100 nM RA for 2 days. By generating deletions in the beta 1-AR promoter, a region between -125 and -100 was found to mediate a 3-fold induction in cells exposed to RA for an additional 2 days. Through site-directed mutagenesis of this region, it was determined that the RA responsive element (RARE) was organized as a direct repeat separated by 5 nucleotides in which the 5'-most AGGTCG half-site was between nucleotides -106 and -101 and the 3'-most AGGTCA half-site was between nucleotides -117 and -112. The RA receptor alpha (RAR alpha) isoform bound to the oligomer representing the sequences between -125 and -100 as a heterodimer complex with the retinoid X receptor alpha (RXR alpha). In a separate study, it was determined that the nucleotides between -125 and -100 are involved in thyroid hormone-mediated activation of the beta 1-AR gene in ventricular myocytes. Therefore, transcriptional activation of the beta 1-AR gene by thyroid hormone or RA involves a single binding site in the promoter.