Negative regulation of the glycoprotein hormone alpha gene promoter by thyroid hormone: mutagenesis of a proximal receptor binding site preserves transcriptional repression.

Transcription of the glycoprotein hormone alpha gene is repressed by the thyroid hormone receptor (TR) in a hormone dependent manner. Previous studies identified a TR binding site immediately downstream of the TATA box. Site directed mutagenesis and transient gene expression studies were used to evaluate the role of this TR binding site as a negative thyroid response element (nTRE). Mutagenesis of the putative negative thyroid response element (nTRE) site eliminated TR binding but failed to eliminate negative regulation by T3. A mutation which converted the putative nTRE to a higher affinity palindromic element did not enhance repression, but rather eliminated thyroid hormone dependent negative regulation. Proximal alpha promoter sequences between -100 and +44 were replaced with a heterologous thymidine kinase promoter resulting in a construct that was not repressed by T3 treatment. This finding confirmed that repression required proximal alpha promoter sequences and also indicated that repression did not occur by interference with the function of upstream the alpha gene enhancers. These studies indicate that TR binding adjacent to the TATA box is not required for T3 mediated repression of the alpha promoter and suggest that negative regulation may involve protein-protein interactions with promoter-specific transcription factors.