EFFECTS OF VARYING THE POSITION OF THYROID-HORMONE RESPONSE ELEMENTS WITHIN THE RAT GROWTH-HORMONE PROMOTER - IMPLICATIONS FOR POSITIVE AND NEGATIVE REGULATION BY 3,5,3'-TRIIODOTHYRONINE

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat BETA-TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 base-pairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat BETA-TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction. Gel mobility shift assays with pure T3R demonstrated binding to all of the elements studied. The rGH T3RE, therefore, functions as a T3-dependent enhancer across a wide range of positions. In the context of the rGH promoter, specific nT3REs reduced T3 induction. The magnitude of the increase or decrease in T3 induction, however, was influenced by the position of the T3RE or nT3RE. We conclude that positive and negative regulation by T3 is dependent on the nature of the binding site, but is influenced by promoter position.