DOMINANT-NEGATIVE ACTIVITY OF AN ENDOGENOUS THYROID-HORMONE RECEPTOR VARIANT (ALPHA-2) IS DUE TO COMPETITION FOR BINDING-SITES ON TARGET GENES

Regulation of development and metabolism by thyroid hormone (T3) may b e influenced by a non-T3 binding T3 receptor (TR) isoform, TRalpha2, w hich can inhibit transcriptional activation by legitimate TRs. Numerou s mechanisms have been postulated to explain the dominant negative act ions of TRalpha2, including competition for target genes, formation of inactive heterodimers, and squelching. We have found that excess TRal pha2 was required to inhibit TRalpha1-mediated transactivation from mu ltiple T3 response elements (TREs). Inhibition of T3 action by TRalpha 2 was specific for TRE-containing genes, because a GAL4/TRalpha1 chime ra, which heterodimerized with the 9-cis-retinoic acid receptor (RXR) and activated transcription from the GALA binding site in the presence of T3, was not inhibited by TRalpha2. In contrast, TRalpha2 inhibited transactivation by TRalpha/VP16, a chimeric protein containing the N- terminal DNA binding domain (DBD) of TRalpha1 fused to the transcripti onal activation domain of VP16. Indeed, TRalpha2 inhibited the binding of TRalpha1 monomers, homodimers, and RXR-heterodimers to DNA in vitr o, whereas the TRalpha2 C terminus alone did not. Although TRalpha2 bo und to TREs with less affinity than TRalpha1, it bound directly to tar get genes in the cell nucleus. Furthermore, a TRalpha2 mutant which bi nds more avidly to TREs was a more effective inhibitor of T3 action th an wild type TRalpha2. Together these data indicate that TRalpha2 inhi bits T3 action by competing for binding to TREs.