Fig. 1. Structure of the DR4 and rGH TRE and Schematic of the Different TR Isoforms A, The nucleotide sequence of a consensus DR4 and the native rGH TRE are shown, with the three hexanucleotide half-site sequences (A–C) highlighted; arrows beneath indicate relative orientation. B, The four predominant TR isoforms are illustrated from N to C terminus. The locations of the DNA binding and hormone binding domains within these receptors are indicated. The TRβ0 and TRβ2 isoforms (TRβ1 and TRβ2 in mammals) are encoded from a single genetic locus by alternative mRNA splicing, and differ only in their amino-terminal A/B domains as indicated. The TRα1 isoform is encoded by a distinct locus and differs from the β isoforms at multiple positions throughout the open reading frame. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 2. All Four TR Isoforms Are Able to Bind as Receptor Dimers to Dual Half-Site TREs The different TR isoforms were obtained from a recombinant baculovirus/Sf9 cell expression system and were tested for the ability to bind to radiolabeled DNA probes using an EMSA protocol. Phosphorimager scans of representative electrophoretograms are presented. A, TRα1 binding to a single half-site (1-HS) vs. a DR4 element was compared in the absence and presence of RXRα, and in the absence or presence of T₃ hormone, as indicated above the panel. The positions of unbound DNA (free probe, fp), TRα1 monomer/DNA complexes (TRα1), TRα1 homodimer/DNA complexes [2(TRα1)], TRα1/RXR heterodimer/DNA complexes (TRα1/RXR), and RXR homodimer/DNA complexes [2(RXRα)] are indicated on the left. Preparations isolated from nonrecombinant baculovirus/SF9 cells were used as negative controls (Non-Recomb). B, TRβ0 binding to the 1-HS and DR4 DNA probes was analyzed by the methods in panel A. C, The ability of the different TR isoforms to bind to a series of two half-site DNA response elements was characterized. See Table 1 for the nucleotide sequences of the probes. The position of monomeric, homodimeric, and heterodimeric protein/DNA complexes are indicated to the left of the panel for each receptor as in panel A. To conserve space, only the relevant portion of each phosphorimager scan, containing the receptor/DNA complexes, is shown. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 5. Trimeric TRβ Binding Requires the Presence of Three Half-Sites Displayed with Appropriate Spacings and Orientations The ability of TRβ0 and TRβ1 to bind to altered versions of the rGH element was tested. A, Disruption of any one half-site in the rGH element prevents trimer binding by TRβ0 or β1. Each reaction contained 50 nm of ³²P-labeled oligonucleotide probe and equal mass amounts of TRα1 and TRβ0. The identity of the DNA response element used in each experiment is depicted above each lane; mut A, mut B, or mut C denote rGH elements with the indicated half-site mutated as to be dysfunctional (see Fig. 1A and Table 1). B, The ability of TRα1 and β0 to bind to a series of different artificial response elements was tested; the effect of altering the orientation of the B and C half-sites was examined. EMSAs were performed as in panel A except the oligonucleotide probes were used at 100 nm. See Table 1 for the nucleotide sequences of the probes. C and D, The effect of different half-site spacings on trimer formation by TRβ0 was examined. EMSAs were performed as in panel B. The receptors used in each analysis are indicated to the left of the panel. E, The ability of TRβ0 and TRα1 to bind to a selection of natural tripartite DNA elements was examined by EMSA as in panel A. See Table 1 for the sequences of the probes. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 4. TRβ0 and TRβ1, But Not TRβ2, Can Form Heterotrimers with All Three RXR Isoforms on the Tripartite rGH TRE The ability of the different TRβ isoforms to heterodimerize with RXRs was examined. A, TRβ0 and β1 can form both heterodimers and heterotrimers on suitably reiterated response elements. EMSA reactions were performed as in Fig. 2, and contained equal amounts of each corresponding TRβ isoform, a series of RXR concentrations (indicated as triangles above the panel), and 100 nm of the ³²P-labeled oligonucleotide probe indicated, as described in Materials and Methods. The TR isoform used in each analysis is indicated to the right of each panel; lanes 18–20 contain the maximum amount of RXR used in the dilution series, but in the absence of TRs. B, Quantification of TRβ0 binding to the DR4 (left panel) and consensus rGH (right panels) elements in the absence or presence of RXRα; the amount of probe migrating as a receptor homodimer or heterodimer is presented; bars represent the mean of four experiments with standard errors. C, The identities of the receptors in the dimeric and trimeric complexes were confirmed by use of antibody supershifts. EMSA antibody supershift experiments were performed as described in Materials and Methods. Antibodies to the different receptors were either omitted from (−) or included in the EMSA reactions (T = anti-TRβ; X = anti-RXR). From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 3. The TRβ Isoforms, But Not TRα, Bind to the rGH TRE as Protein Trimers The ability of different TR isoforms to bind to a DR4 element vs. an rGH response element was analyzed. A, DNA binding by TRα1 and TRβ0 was compared. EMSAs were performed using 50 nm of ³²P-labeled oligonucleotide probes representing a DR4 or rGH TRE, and identical ranges of TRα1 or TRβ0 concentrations, as indicated above the panels and as described in Materials and Methods. A phosphorimager scan of a representative electrophoretogram is shown. The positions of receptor/DNA complexes representing TRα1 homodimers, TRβ0 homodimers, and TRβ0 homotrimers are indicated on the sides of the panel (see legend), as is the position of free DNA probe not bound to protein (fp). B, DNA binding by the three different TRβ isoforms was compared. The EMSA was performed as in panel A except 100 nm of each oligonucleotide probe was used. See Table 1 for the nucleotide sequences of the probes. A phosphorimager scan of a representative electrophoretogram is provided. For the TRβ2 experiment in panel B, the gain in the phosphorimager was elevated 4-fold compared with TRβ0 and TRβ1 to permit the weak formation of trimeric complexes by this isoform to be detected. C, The ability of the TRβ isoforms to bind to the DR4 element as dimers or to the consensus rGH element as trimers was compared as in panel B and quantified. Bars represent the mean of four experiments with standard errors. TRβ0 binding to the DR4 element was defined as 100. D, TRβ0 binding to a DR4, native rGH, consensus rGH, and mutated versions of the native rGH element was compared by EMSA with phosphorimager analysis and quantified. Reactions contained 100 nm of each oligonucleotide probe and a range of TRβ0 concentrations. Only the highest two TRβ0 concentrations were used for the A–C site mutants. The amount of receptor/DNA complexes migrating as a receptor homodimer (top panel), or as a receptor homotrimer (bottom panel) are presented; maximal TRβ0 binding to the DR4 and con rGH elements was defined as 100. The average of four independent experiments is presented; error bars represent se. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 7. TRβ0 Activates Transcription from an rGH Element More Strongly than Does TRβ2, Whereas Both Receptors Activate From a DR4 Element Equally The ability of TRβ0 and of TRβ2 to activate transcription through DR4 or rGH response elements was tested by a cell transfection protocol, as described in Materials and Methods. A, The ability of TRβ0 and of β2 to activate transcription was tested over a range of receptor concentrations. CV-1 cells were transfected with a luciferase reporter gene bearing either a DR4 or native rGH TRE element, and a range of concentrations of pSG5 vectors expressing either TRβ0 or TRβ2, as indicated. A pSG5 empty construct was added to maintain total DNA concentration constant. All transfections were analyzed in the presence of 100 nm T₃, and a β-galactosidase expression vector was included as an internal control for transcription efficiency. Fold relative activation was calculated as the ratio of reporter gene expression in the presence vs. the absence of a TR allele in the pSG5 expression vector; the mean fold relative luciferase values of four independent experiments are shown. Error bars represent the standard error. The results for TRβ0 (square symbols) and TRβ2 (triangles), either minus (open symbols) or plus (solid symbols) 100 nm T₃ are shown. B, Transient transfections were performed as in panel A, except 5 ng of the pSG5-TR vector was tested with the different TRE luciferase reporters indicated. The A, B, and C site mutations were in the context of the native rGH element. The results for TRβ0, plus T₃ (black bars) and TRβ2 plus T₃ (open bars) are shown. Results represent the mean of four independent experiments; error bars are the standard error. C, The same experiments as in panel B are shown, but in the absence of hormone. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 6. Trimeric TRβ0 Complexes Assembled on the rGH Element Can Interact with Corepressors and Coactivators The ability of TRβ0 dimeric and trimeric complexes (formed on the DR4 or consensus rGH element, respectively) to interact with corepressors and coactivators was analyzed. GST-fusion proteins containing the receptor interaction domains of SMRT, of N-CoR, or of ACTR were added to the EMSA binding reactions in the presence or absence of 1 μm T₃ as indicated above the panels. Interaction of the corepressor or coactivator with the receptor/DNA complex leads to formation of a complex with slower mobility than that of receptor/DNA alone (supershift). All lanes contained 100 nm³²P-labeled oligonucleotide probe and either TRβ0 (top panel) or TRβ0/RXRα (bottom panel); receptor concentrations were identical in all lanes of each panel. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society

Fig. 8. TRβ0 Can Bind an Expanded Segment of the rGH Promoter as a Presumptive Receptor Pentamer A, The nucleotide sequence and arrangement of possible half-sites in the regions encompassing and flanking the core rGH element is presented (29 ). Each presumptive half-site is underlined. Half-sites A–C comprise the trimeric rGH TRE previously noted in Fig. 1. B, TRβ0 can form both homo and heteropentameric receptor complexes on the extended rGH promoter element. For each binding reaction, TRβ0, or a mix of TRβ0 and RXRβ, was incubated with 100 nm radiolabeled oligonucleotide probe (representing a DR4, rGH, or rGH 5-half-site TRE) and the resulting complexes were resolved by EMSA as in Fig. 2 and Materials and Methods. A phosphorimager scan of a representative electrophoretogram is presented. From: Novel Mode of Deoxyribonucleic Acid Recognition by Thyroid Hormone Receptors: Thyroid Hormone Receptor β-Isoforms Can Bind as Trimers to Natural Response Elements Comprised of Reiterated Half-Sites Mol Endocrinol. 2005;19(1):35-51. doi:10.1210/me.2003-0289 Mol Endocrinol | Copyright © 2005 by The Endocrine Society