Nonbiased identification of DNA sequences that bind thyroid hormone receptor alpha 1 with high affinity.
Thyroid hormone receptors are transcription factors that bind to specific DNA sequences and regulate gene expression in a ligand-dependent manner. Although thyroid hormone receptors are known to bind to the hexamer 5'-AGGTCA, it is not known if this represents the optimal binding site. Therefore, a nonbiased strategy was used to identify DNA sequences which bind thyroid hormone receptor alpha 1 with high affinity. Such DNA sequences were isolated from a pool of random sequences using a strategy combining an electrophoretic mobility shift assay with the polymerase chain reaction. It was found that thyroid hormone receptor alpha 1 binds with highest affinity to the octamer 5'-TAAGGTCA. Mutation of the two 5'-nucleotides decreased the affinity of thyroid hormone receptor alpha 1 for this DNA sequence approximately 5-fold, and the importance of those nucleotides in receptor binding was confirmed by DNA footprinting. A single copy of the octamer sequence (but not the hexamer 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 alpha 1 is 2 base pairs larger than previously thought, and that a single binding site can function as a response element. In addition, we speculate that the optimal binding sites for thyroid hormone, vitamin D, and retinoic acid receptors may not be identical, as had previously been thought.
Katz
R W
RW
Endocrinology Division, University of Michigan Medical Center, Ann Arbor 48109-0678.
Koenig
R J
RJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D011817:Rabbits; D011988:Receptors, Thyroid Hormone / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology
D009838:Oligodeoxyribonucleotides; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D014157:Transcription Factors; D014284:Triiodothyronine; D004247:DNA; D008715:Methionine; D015500:Chloramphenicol O-Acetyltransferase