THE LIGAND-BINDING DOMAINS OF THE THYROID-HORMONE RETINOID RECEPTOR GENE SUBFAMILY FUNCTION IN-VIVO TO MEDIATE HETERODIMERIZATION, GENE SILENCING, AND TRANSACTIVATION

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gen e subfamily contain a series of heptad motifs important for dimeric in teractions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RAR s and retinoid X receptors [RXRs]), the 1,25 dihydroxyvitamin D-3 rece ptor (VDR), and the receptors that modulate the peroxisomal beta-oxida tion pathway (PPARs). These receptors bind to their DNA response eleme nts in vitro as heterodimers with the RXRs. Unliganded receptors in vi vo, in particular the T3Rs, can mediate gene silencing and ligand conv erts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4 -RXR chimera containing the yeast GAIA DNA-binding domain and the LED of RXR beta. GAL4-RXR activates transcription from GAL4 response eleme nts in the presence of 9-cis RA. Unliganded T3R, which does not bind o r activate GAL4 elements, represses the activation of GAL4-RXR by 9-ci s RA in HeLa cells. However, addition of T3 alone leads to transcripti onal activation. These findings suggest that T3R can repress or activa te transcription while tethered to the LED of GAL4-RXR and that hetero dimerization can occur in vivo without stabilization by hormone respon se elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LED of RXR be ta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is rep ressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad of T3R is important for gene silencing and that the LED of RXR does not exhi bit silencing activity. This conclusion was verified with GAL4-LBD chi meras and with wild-type receptors in assays using appropriate respons e elements. These studies indicate that the LED has diverse functional roles in gene regulation.