Thyroid hormones and their receptors in transcriptional regulation

The thyroid hormone receptors (TRs) are encoded by two genes, alpha and ß, and belong to a family of hormone activated nuclear receptors. This family includes the receptors for retinoids and vitamin D3 as well as the receptors for steroid hormones. Transcriptional control by hormone is mediated through receptor binding to target gene DNA and subsequent control of gene regulation. Here we have studied several aspects of gene regulation controlled by TR and its oncogenic counterpart P75gag-v-erbA, to gain insights into the specificity in DNA-binding and into which domains of the receptor are important for intracellular localization and control of erythroid differentiation.

The specificity of binding of TR to DNA regulatory response elements (TREs) was tested with band shift analysis in the absence and presence of thyroid hormones. TR interacts as a monomer or homodimer to form complexes with direct repeat, palindromic and with everted repeat AGGTCA core motifs. The thyroid hormones T3 and T4, but not T2, disrupt homodimer- and increase monomer complexes bound to TREs. A TR conformational change induced by T3 is detected in TR monomers and in TR which forms a heterodimer with RXR.

TR is unique within the superfamily of receptors by the ability to bind response elements with core motifs positioned in several different configurations. TR binding to direct repeat and palindromic TREs containing O to 6 nucleotides between the core motifs (spacer) was assayed in band shift analyses. High affinity binding and low off rate was observed for complexes containing TR on TREs with direct repeats separated by a spacer of 4 nucleotides and on a palindromic TRE without a spacer. The oncoprotein P75gag-v-erbA blocks TR-induced erythroid differentiation. A number of TR / P75gag-v-erbA chimeric proteins were tested for their effects of differentiation and it was shown that the DNA binding domain (DBD) of P75gag-v-erbA is essential for self-renewal and to keep cells undifferentiated.

TR is localized to the nucleus both in the presence and absence of thyroid hormones. To define the regions important for nuclear localization we determined the intracellular localization of TR, P75gag-v-erbA and various N-terminal variants of TR by immunocytochemistry following transfection. The data show that the N-terminal first 12 amino acids of TR are essential for exclusive nuclear localization. P75gag-v-erbA represses TR/T3 activated transcription. To pinpoint which regions in P75gag-v-erbA that are responsible for this, we expressed P75gag-v-erbA and various TR / P75gag-v-erbA chimeric receptors in cells expressing high levels of endogenous TR. A chimeric receptor, containing the DBD and the N-terminus from TR and the ligand binding domain from P75gag-v-erbA, was able to repress a TRE not regulated by P75gag-v-erbA.