NONBIASED IDENTIFICATION OF DNA-SEQUENCES THAT BIND THYROID-HORMONE RECEPTOR-ALPHA-1 WITH HIGH-AFFINITY

Thyroid hormone receptors are transcription factors that bind to speci fic DNA sequences and regulate gene expression in a ligand-dependent m anner. Although thyroid hormone receptors are known to bind to the hex amer 5'-AGGTCA, it is not known if this represents the optimal binding site. Therefore, a nonbiased strategy was used to identify DNA sequen ces which bind thyroid hormone receptor alpha1 with high affinity. Suc h DNA sequences were isolated from a pool of random sequences using a strategy combining an electrophoretic mobility shift assay with the po lymerase chain reaction. It was found that thyroid hormone receptor al pha1 binds with highest affinity to the octamer 5'-TAAGGTCA. Mutation of the two 5'-nucleotides decreased the affinity of thyroid hormone re ceptor alpha1 for this DNA sequence approximately 5-fold, and the impo rtance of those nucleotides in receptor binding was confirmed by DNA f ootprinting. A single copy of the octamer sequence (but not the hexame r AGGTCA) could impart T3 responsiveness to a heterologous promoter in a transient transfection assay. The results indicate that the optimal binding site for thyroid hormone receptor alpha1 is 2 base pairs larg er than previously thought, and that a single binding site can functio n as a response element. In addition, we speculate that the optimal bi nding sites for thyroid hormone, vitamin D, and retinoic acid receptor s may not be identical, as had previously been thought.