Identification of Direct Repeat 4 as a Positive Regulatory Element for the Human TR4 Orphan Receptor: A MODULATOR FOR THE THYROID HORMONE TARGET GENES

Yi-Fen Lee,Huei-Ju Pan,J Peter H Burbach,Eugene Morkin,Chawnshang Chang

doi:10.1074/jbc.272.18.12215

Abstract

While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct repeat 1-hormone response element (DR1-HRE) and DR2-HRE, we now report that TR4 can also induce the transcriptional activity of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay. Electrophoretic mobility shift assay and Scatchard analysis reveal a strong binding affinity (dissociation constant = 2 nM) between TR4 and DR4-HRE. The induction mediated by TR4 was detected not only in the synthetic DR4-HRE but also in some genes, such as rat alpha-myosin heavy-chain and S14 genes, containing the DR4 or DR4-like motif, which have been suggested to be the response elements for a thyroid hormone receptor. Our data also demonstrate this TR4-mediated gene induction is TR4 dose- and DR4 sequence-dependent. Together, our data suggest that DR4-HRE can be a positive regulatory element for TR4, which may be able to induce the transcriptional activity of the genes containing such positive HREs.

Highlights

While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct repeat 1-hormone response element (DR1HRE) and DR2-hormone response elements (HREs), we report that TR4 can induce the transcriptional activity of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay
The binding specificity was further confirmed by adding excess unlabeled DR4-HRE or mutated DR4 (Fig. 1A)
Like the TR2 orphan receptor, TR4 can serve as a repressor in RAR/RXR-mediated gene induction and transcription from the major late promoter of the SV40ϩ55 gene [16, 22, 28]

Summary

Introduction

While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct repeat 1-hormone response element (DR1HRE) and DR2-HRE, we report that TR4 can induce the transcriptional activity of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay. Our data suggest that DR4-HRE can be a positive regulatory element for TR4, which may be able to induce the transcriptional activity of the genes containing such positive HREs. The nuclear receptor superfamily comprises a large group of ligand-dependent transcriptional factors that control the expression of target genes by binding to their cognate hormone response elements (HREs)1 [1,2,3,4]. 1 The abbreviations used are: HRE, hormone response element; TR4, TR4 orphan receptor; DR, direct repeat; ␣-MHC, ␣-myosin heavy chain; FUR, far upstream regulatory region; HIV, long terminal repeat of the human immunodeficiency virus I; T3R, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; RA, retinoic acid; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase. Our previous data suggested that TR4 preferred to bind to the HREs, which consist of two DRs of consensus half-site, rather than a palindrome. TR4 can repress the retinoic acid-induced transactivation by competitive DNA binding to the response elements of other receptors (i.e. DR5 for retinoic acid receptor (RAR) and DR1 for retinoid X receptor (RXR)). In addition, the TR4-mediated suppression can be found in the gene promoter containing DR2-like response element that is located in the ϩ55 region of the SV40 major late promoter [16]

Methods

Results

Conclusion