INTERACTION BETWEEN THE THYROID-HORMONE RECEPTOR AND COFACTORS ON THE PROMOTER OF THE GENE ENCODING PHOSPHOENOLPYRUVATE CARBOXYKINASE

Using transient-transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phosphoenol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at position -324 to -297 was protected by the thyroid-hormone receptor. This receptor was overexpressed in HeLa cells using a vaccinia virus expression vector. DNA-binding assays with this receptor-enriched nuclear HeLa cell extract revealed that only 20% of the thyroid hormone receptor was able to bind the target-sequence with high affinity (4 nM). Titration with nuclear extract from hepatocytes increased the percentage of c-erbA molecules able to bind to this thyroid hormone-responsive element 4-fold without a change in affinity. Our data show that the complex of the thyroid hormone responsive element of the promoter of the phosphoenolpyruvate carboxykinase gene and the thyroid hormone receptor contains only a single receptor molecule suggesting the formation of a heterodimer complex. Accordingly, this thyroid hormone receptor/DNA complex is formed only in the presence of co-factors that are present in limiting amounts in the hepatocyte nucleus.