Thyroid hormones and their receptors in transcriptional regulation

Abstract: Thyroid hormones and their receptors in transcriptional regulation Monika Andersson From the Department of Cell and Molecular Biology The Medical Nobel Institute Karolinska Institutet S-17177 Stockholm, Sweden 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 includesthe receptors for retinoids and vitamin D3 as well as the receptors for steroid hormones.Transcriptional control by hormone is mediated through receptor binding to targetgene DNA and subsequent control of gene regulation. Here we have studied several aspects of gene regulation controlled by TR and itsoncogenic counterpart P75gag-v-erbA, to gain insights into the specificity in DNAbinding and into which domains of the receptor are important for intracellular localizationand control of erythroid differentiation. The specificity of binding of TR to DNA regulatory response elements (TREs) wastested 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, palindromicand with everted repeat AGGTCA core motifs. The thyroid hormones T3 and T4, but notT2, disrupt homodimer- and increase monomer complexes bound to TREs. A TR conformationalchange induced by T3 is detected in TR monomers and in TR which forms a heterodimerwith RXR. TR is unique within the superfamily of receptors by the ability to bind responseelements with core motifs positioned in several different configurations. TR bindingto direct repeat and palindromic TREs containing O to 6 nucleotides between the coremotifs (spacer) was assayed in band shift analyses. High affinity binding and lowoff rate was observed for complexes containing TR on TREs with direct repeats separatedby a spacer of 4 nucleotides and on a palindromic TRE without a spacer. The oncoprotein P75gag-v-erbA blocks TR-induced erythroid differentiation. A numberof TR / P75gag-v-erbA chimeric proteins were tested for their effects of differentiationand it was shown that the DNA binding domain (DBD) of P75gag-v-erbA is essentialfor 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 intracellularlocalization of TR, P75gag-v-erbA and various N-terminal variants of TR by immunocytochemistryfollowing transfection. The data show that the N-terminal first 12 amino acids ofTR are essential for exclusive nuclear localization. P75gag-v-erbA represses TR/T3 activated transcription. To pinpoint which regionsin P75gag-v-erbA that are responsible for this, we expressed P75gag-v-erbA and variousTR / P75gag-v-erbA chimeric receptors in cells expressing high levels of endogenousTR. A chimeric receptor, containing the DBD and the N-terminus from TR and the ligandbinding domain from P75gag-v-erbA, was able to repress a TRE not regulated by P75gag-v-erbA. Keywords: Thyroid hormone receptor, regulatory response element, palindrome, directrepeat, everted repeat, DNA hinding domain, P75gag-v-erbA, differentiation, nuclearlocalization, repression. Stockholm 1997 ISBN 9 1-628-2744-8