Abstract
T3 (triiodothyronine) response elements (TREs) consist of pairs of strong and weak (S and W), 10-nucleotide T3 receptor (TR) monomer binding sites (half-sites). We report that the number and order of S and W half-sites in a direct repeat TRE determines whether it mediates ligand-dependent or independent transcriptional activation or inhibition in the presence of TR or TR and 9-cis-retinoic acid receptor (RXR); and whether a TRE is preferentially bound by TR homodimers, TR-RXR heterodimers, or CV1 cell TR accessory protein (TRAP)-TR heterodimers. TR homodimers bound equally to TREs composed of the 5'-S and 3'-W (SW) and the opposite (WS) arrangement of half-sites. TR-RXR gamma heterodimers bound SW better than WS. TR-TRAP heterodimers bound WS better than SW. Transcription of a reporter gene cis-linked to WS responded to unliganded TR and RXR, and either ligand stimulated expression 2-fold more. Reporter expression cis-linked to SW was not altered by unliganded receptors, and T3 stimulated transcription only in the presence of both TR and RXR. SS was strongly activated by liganded, but not by unliganded TR. SS was activated by unliganded TR and RXR gamma together, and T3 further stimulated transcription 2-fold. Under these conditions, transcription was inhibited 60% by 9-cis-retinoic acid.
Highlights
R X R y, 9-cis-RA reduced CAT expression by 60%,whether or pressing both Ts receptor (TR) and R X R y,the responses observed are likely not T3 was present. These results demonstrate thSatS-CAT is due mainly to theTR-RXRy heterodimer, since overexpression positively responsive to T3, and negatively responsive to 9 4 s - of R X R y probably minimizes the formation of TR-TRAP het
Receptor binding and transcriptional transactivation do not always conform to the 3, 4, 5-rule,which proposes that 1,25-dihydroxyvitaminD3receptor, TR, and RAR bind and transactivate DR TREs in which the core hexanucleotide consensus sequenceAGGTCA is separated by [3, 4], or 5 nucleotides, respectively (15).First, the binding specificity of at least RARs and RXRs do not correspond to these rules(18).Second, the analysisused to formulate th3e,4, 5-rule did not considerthe role of nucleotides flanking thecore consensus element on receptor binding
At least in thecase of TR, nucleotides flanking the core consensus sequence have a major role in determining receptor binding strength ( 1 6, 19, 20)
Summary
Determined by the Number and Order of High AffinityHalf-sites in a T3Response Element*. The receptors for T3’ (TRs), all-trans-retinoic acid (RARs), pairs, while RXRs bind response elements inwhich DR motifs. Press the transcriptioonf specific genes by binding as dimers to We have conducted a systematic study of the effects of TRE small cis-linked DNA sequences termed response elements. The abbreviationsused are: T,, triiodothyronine; TRE,T, response element; TR, T, receptor; RAR, all-trans-retinoicacid receptor; RXR, 9-cis-retinoic acid receptor;TRAP, TR-associated protein; DR, direct repeats; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; rGH, rat growth hormone; TREpal, palindromic TRE; S, strong;W, weak; 9-cis-FU, 9-cis-retinoic acid. 7 pg of nuclear rect repeatof the 312 and 311half-sites of the rGH intronic TRE, spacepdroteinwasutilizedin EMSAs. Preliminarystudiesindicated that by four base pairs,5'-GATCTCTGAGGTAACTTGAGGCTGAGA-3a'nd maximum enhancement of specific TR binding was obtained with this.
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