REQUIREMENT OF REGULATORY ELEMENTS FOR THYROID HORMONE (TH) RECEPTOR (TR)alpha2 INHIBITION OF TH-TRbeta1 DEPENDENT TRANSACTIVATION

In this report we provide evidence that the inhibition of TH-TR?1 transactivation mediated by TR?2 does not require binding of this non-hormone binding TR variant to the response elemenent and propose an alternative mechanism involving basal transcription factors. An attempt has been made to investigate this hypothesis using two native TH-regulated genes: the brain-specific myelin basic protein (MBP) gene, with a TATA-containing promoter and the malic enzyme (ME), coding for a housekeeping gene characterized by the presence of a GC-rich region close to the transcriptional start site; structure of TREs differ in these two genes by the presence of an inverted palindromic sequence or a direct repeat, respectively. Inhibitory effect of TR?2 was first evaluated in transient transfection assays in NIH3T3 cells using MBP and ME native promoter-CAT reporters cotransfected with TR?1 and TR?2 expression vectors and cultured in the presence of TH. Three-fold excess of TR?2 expression plasmid completely abrogated TH-TR?1-mediated transactivation of the MBP, but not ME promoter. Since in gel mobility shift assays TR?2 failed to interact with the wild type ME-TRE, mutations were introduced to recover TR?2 binding without affecting TH-dependent activation. However, this did not facilitate TR?2 inhibition of TH-TR?1-mediated transactivation. Replacing the MBP-TRE with ME-TRE in the context of MBP promoter or exchanging the MBP TATA box with the GC rich element present in the initiation site of ME promoter rendered TR?2 inactive as did the introduction of MBP-TRE or TATA-containing regions of MBP in the context of ME promoter. These results suggest that the exchange of a single regulatory element was not sufficient to mediate the TR?2 inhibition. However, simultaneous introduction of MBP-TRE and MBP TATA box in the context of ME promoter appeared to trigger TR?2 inhibitory effect of TH-TR?1-mediated transactivation. In conclusion, our data indicate that inhibition of TR?2 is achieved with a TRE arranged as an inverted palindrome in combination with a TATA box element and binding of TR?2 to a TRE alone is not sufficient for TR?2 inhibition of the TH response. A.F. and J.L. contributed equally to this study.