A method for efficient production of recombinant thyroid hormone receptors reveals that receptor homodimer-DNA binding is enhanced by the coactivator TIF2.
Thyroid hormone receptors (TRs) are ligand-activated transcription factors that mediate the biological effects of thyroid hormone (T3) by binding to thyroid hormone response elements (TREs), typically located in the promoter regions of T3-responsive genes. It is generally held that T3-induced gene activation is mediated by retinoid X receptor (RXR)-TR heterodimers. Although TR homodimers can bind to some TREs, T3 destabilizes this interaction, calling into question the ability of TR to activate transcription in the absence of RXR. TR-DNA binding has been difficult to study in vitro because mammalian TRs are notoriously difficult to produce in Escherichia coli. We considered that this may be due to codon bias. Therefore, we produced TRbeta1 in E. coli Rosetta 2(DE3) which contains a plasmid that overexpresses the tRNAs corresponding to the seven rarest E. coli codons. This resulted in an improved yield of full-length TRbeta1, which we then used in electrophoretic mobility shift assays. We found the coactivator TIF2 greatly enhances binding of T3-occupied TRs to a subset of TREs, suggesting TRs may activate transcription from these TREs in an RXR-independent manner.
Diallo
Ericka M
EM
Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109-0678, USA.
Thompson
Deborah L
DL
Koenig
Ronald J
RJ
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008722:Methods; D051958:Nuclear Receptor Coactivator 2; D011485:Protein Binding; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins; D020218:Response Elements; D047490:Retinoid X Receptor alpha; D037042:Thyroid Hormone Receptors beta; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D014284:Triiodothyronine / Q000378:metabolism
C498001:NCOA2 protein, human; C498003:Ncoa2 protein, rat; D051958:Nuclear Receptor Coactivator 2; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D047490:Retinoid X Receptor alpha; D037042:Thyroid Hormone Receptors beta; D014157:Transcription Factors; D014284:Triiodothyronine; D004247:DNA