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

Abstract

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 α-subunit and rat βTSH genes. The βT₃REs were placed in the background of the wildtype 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 basepairs 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 α-subunit gene in the same position reduced induction. Negative T₃REs from rat βTSH and human a-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.