Nuclear Corepressors Enhance the Dominant Negative Activity of Mutant Receptors That Cause Resistance to Thyroid Hormone

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society Vol. 139, No. 2 Printed in U.S.A. Nuclear Corepressors Enhance the Dominant Negative Activity of Mutant Receptors That Cause Resistance to Thyroid Hormone* TETSUYA TAGAMI AND J. LARRY JAMESON Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611 ABSTRACT The syndrome of resistance to thyroid hormone (RTH) is caused by multiple distinct mutations in the ligand-binding domain of the thyroid hormone receptor-b (TRb). Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Recently, a group of transcriptional cofactors, referred to as corepressors (CoRs), was shown to induce ligand-independent silencing of genes that contain positive T3 response elements. CoRs also play a role in the ligandindependent basal activation of genes that are negatively regulated in response to T3. We hypothesized that CoR might play a role in the dominant negative inhibition by TRb mutants that cause RTH. In gel mobility shift assays, RTH mutants retained interactions with CoRs even in the presence of T3, whereas the ligand dissociated CoR from wild-type TRb. Using Gal4-TR chimeric receptors and a VP16-CoR fusion protein in an interaction assay, a strong positive correlation was found between mutant receptor interactions with CoR and transcriptional silencing activity. A mutation (P214R) that impairs CoR interactions with TR was introduced into the RTH mutants to assess the role of CoR in dominant negative activity. In transient transfection assays, introduction of the P214R CoR mutation decreased RTH mutant silencing of positively regulated genes and basal activation of negatively regulated genes. The dominant negative activity of several different RTH mutants, studied by cotransfection with wild-type receptor, was greatly diminished by the CoR mutation, and this effect was seen with both positively and negatively regulated genes. These results suggest that CoR interactions play a critical role in the dominant negative effect of RTH mutants and support the idea that these proteins are involved in the regulation of genes that are positively as well as negatively regulated by T3. (Endocrinology 139: 640 – 650, 1998) T (amino acids 211–240) in the hinge region of human TRb was shown to cause the loss of basal activation of negatively regulated genes as well as silencing of positively regulated genes (16). This finding suggests that CoRs also play a role in basal activation of genes that are negatively regulated in response to T3. Resistance to thyroid hormone (RTH) is an autosomal dominant disorder that is caused by mutations in the TRb gene (17, 18). Most TRb mutations reduce binding to T3, although some appear to impair transcriptional activity despite near-normal T3 binding (19 –21). Consistent with the dominant mode of transmission, the mutant receptors interfere with the function of normal TRs by a dominant negative mechanism (22–24). Although the mechanism of dominant negative activity is still being investigated, most data support the idea that mutant receptors retain the ability to bind to DNA and block access of normal TRs to their target genes (25). Although hormone resistance occurs to varying degrees in all tissues, the diagnosis of RTH is based primarily upon abnormalities in the TRH-TSH-T3 axis (26). Specifically, RTH is characterized by elevated levels of free thyroid hormone without evidence of appropriate suppression of TSH. The degree of hypothalamic-pituitary resistance establishes a setpoint that defines the circulating hormone levels that act on all other tissues. For these reasons, it is of great interest to examine negatively regulated as well as positively regulated genes when considering the targets for the dominant negative effects of mutant receptors. HYROID hormone receptors (TRs) function as ligandregulated transcription factors that increase or decrease the expression of target genes (1, 2). In the unliganded state, TRs suppress or silence the basal activity of promoters that contain positively regulated hormone response elements (3–5). The addition of ligand (T3) reverses silencing and stimulates transcription to a level that is even greater than the original basal state. In contrast, the basal activity of negatively regulated genes is stimulated by unliganded receptor, and transcription is repressed after the addition of T3 (6 – 8). Recently, nuclear corepressors (CoRs), variously termed nuclear receptor corepressor (NCoR) (9, 10), silencing mediator for retinoid and thyroid hormone receptors (SMRT) (11), thyroid receptor-associating cofactors (12), and thyroid receptor-interacting proteins (13), have been identified. The CoRs interact with the ligand-binding domain (LBD) of nuclear receptors and mediate ligand-independent repression. Several mutations in the so-called CoR box at the aminoterminal end of the LBD disrupt binding to CoRs (9 –11), and there is evidence for additional interactions with more carboxyl-terminal regions of the LBD (14, 15). In a previous study, a mutation (P214R) that corresponds to the CoR box Received July 2, 1997. Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15– 709, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: . * This work was supported by NIH Grant DK-42144. 640 THYROID HORMONE RESISTANCE AND COREPRESSORS 641 Because CoRs play an important role in the function of unliganded TRs and are dissociated upon the addition of T3, we hypothesized that interactions with CoRs might be important for the dominant negative activity of RTH mutants. In this report, we analyzed the effects of a CoR mutant of TRb (P214R) on the dominant negative effect of RTH mutants using positively and negatively regulated T3-responsive promoters. Materials and Methods Plasmid construction and receptor mutagenesis The plasmid PAL-TK-Luc contains two copies of a palindromic thyroid hormone response element (TRE; 59-gatctcAGGTCATGACCTgagatc-39) upstream of the thymidine kinase promoter (TK109) in the pA3 luciferase (Luc) vector (27). DR4-SV40-Luc contains four copies of a direct repeat TRE (59-agcttcAGGTCActtcAGGTCActcga-39) upstream of simian virus 40 (SV40) promoter in the pGL3 Luc vector (Promega, Madison, WI). TSHa-Luc contains 846 bp of the 59-flanking sequence and 44 bp of exon I from the human glycoprotein hormone a-subunit gene in pA3-Luc (28). TSHb-Luc contains 128 bp of the 59-flanking sequence and 37 bp of exon I from the human TSH b-subunit gene in pA3-Luc (16). The pCMV-TEF expression vector (thyrotroph embryonic factor) (29) was provided by M. G. Rosenfeld (University of California, San Diego, CA). The Gal4 reporter plasmid, UAS-TK (...truncated)