Abstract
Thyroid hormone receptors bind to thyroid hormone response elements (TREs) as heterodimers with 3,5,3'-L-triiodothyronine (T3) receptor auxiliary protein (TRAP) and retinoid X receptors (RXRs). Currently, it is not known whether TR/TRAP or TR/RXR heterodimers need to bind to both TRE half-sites and whether there is a preferred orientation for TR/RXR heterodimer binding to TREs or transcriptional activation. Accordingly, we created a mutant TR alpha (TR-P box) by changing 3 amino acids in the P box region of the first zinc finger of the DNA-binding domain to that of the glucocorticoid receptor (GR), and we examined wild-type TR alpha and TR-P box complex binding to hybrid response elements containing TRE and glucocorticoid receptor element (GRE) half-sites arranged as a direct repeat with a four-nucleotide gap. TR-P box/RXR heterodimers selectively bound to the hybrid response elements in which GRE half-site was the downstream half-site, whereas TR alpha/RXR bound to hybrid response elements in which GREs were in either position. Additionally, TR/TRAP or TR/RXR heterodimer required two half-sites for binding to DNA, with strong binding to at least one of the half-sites. Last, co-transfection assays and methylation interference studies using the hybrid response elements suggest that the sequential arrangement of strong and weak half-sites in the TRE may be a critical determinant of TR/RXR heterodimer binding and transcriptional activation.
Highlights
Half-site Arrangementof Hybrid Glucocorticoid and Thyroid Hormone Response ElementsSpecifies Thyroid Hormone Receptor Complex Bindingto DNA and Transcriptional Activity*
We found that the sequential arrangementof strong (TRE) and weak (GRE) halfsites in the thyroidhormoneresponse elements (TREs) may be a critical determinant for TWRXR heterodimer binding to theTRE and transcriptionalactivation
We first analyzed Thyroid hormone receptors (TRs)-P box binding to hybrid response elements containing either TRE or GRE half-sites arranged as a direct repeatwith a four nucleotide gap sequence (Table I)
Summary
G. Rosenfeld, were confirmed by DNA sequencing. Each cDNA was linearized with University of California, San Diego), and 150,000cpm of methylated the appropriate restriction endonuclease and used as a template for labeled probe were performed and the heterodimer bands andprofbreee. The DNA was isolated, treated wi1thM piperidine for 30 thionine-labeled receptors were produced from rabbit reticulocyte min at 90 "C, purified, lyophilized, and suspended in formamide loading lysates according tothe manufacturer's instructions(Life Technologies, buffer, before being subjected to electrophoresiosn 8%denaturing poly-. Co-transfection Studies-GG, GT, TG, and Tl'oligonucleotides containing HindIII andXhoI restriction sitoens 5'and 3'ends werecloned proteins of expected molecular weights. Both rTRa and TR-P box pro- intothe PT109 vector(whichcontainsaherpessimplexthymidine teins had similar binding to['"I]T,.
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