ABSTRACT
Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator. The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation. To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase. Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed. Final yields were approximately 1 mg/liter of culture. Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM. An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor. Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence. This preparation will be useful in exploring the mechanisms of receptor activity.
Subject(s)
Receptors, Thyroid Hormone/genetics , Base Sequence , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Glutathione Transferase/genetics , Humans , Molecular Sequence Data , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/isolation & purification , Receptors, Thyroid Hormone/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Transcription Factors/metabolism , Triiodothyronine/metabolismABSTRACT
Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.
Subject(s)
DNA/metabolism , ErbB Receptors/genetics , Promoter Regions, Genetic , Receptors, Retinoic Acid , Receptors, Thyroid Hormone/metabolism , Sp1 Transcription Factor/metabolism , Transcription Factors , Transcription, Genetic , Animals , Base Sequence , Binding, Competitive , Cells, Cultured , Drosophila , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Receptors, Cell Surface/metabolism , Retinoid X ReceptorsABSTRACT
The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.
