Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo
Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford
Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford
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Article Endocrinology

Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo

Abstract

Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-β was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-β DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-βGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-β KO (TR-β–/–) mice. However, TR-βGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-β–/– mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.

Authors

Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford

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Figure 1

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Generation of TR-βGS/GS and TR-β–/– mice. Schematic strategy of homologo...
Generation of TR-βGS/GS and TR-β–/– mice. Schematic strategy of homologous recombination in TR-βGS/GS (a) and TR-β–/– mice (b) is illustrated. Diagrams show the WT TR-β locus, the targeting vectors, ES-targeted alleles, and the F1 mutant alleles after the ACN cassette is excised. The mutated exon 3 is shown as shaded boxes. H, Hind III; K, Kpn I; RV, Eco RV; B, Bgl II; X, Xba I. The locations of ES, GS, and KO probes for Southern blot analysis are indicated. The ACN cassette is flanked by loxP sites indicated by black arrowheads. Arrows indicate the positions of PCR primers (5′, match/mismatch, KO5′, KO3′) used for DNA genotyping. The site of restriction fragments obtained by Southern blot analysis is given in kb. Chi, chimeric. (c) Southern blot analysis of DNA from ES clones. WT (11.5 kb) and targeted (8.0 kb) Hind III alleles were detected by a 3′ external ES probe. (d) Southern blot analysis of DNA from F2 mice resulting from a GS125 KI heterozygous intercross compared with chimeric animals. After Eco RV digestion, a 4.8-kb band was detected in targeted allele of chimeric mice using GS probe. After self-excision of ACN cassette, a 3.8-kb band is obtained from the mutant allele. (e) Genotyping of F2 KI offspring by mismatch PCR. The WT allele was detected with WT match primer set and mutant allele was detected only with the mismatch (mutant) primer set. (f) Southern blot analysis of TR-β KO F2 siblings using the KO probe. The mutant allele demonstrated a longer (7.8 kb) band after Kpn I digestion versus the WT allele of 3.8 kb. (g) PCR genotyping of F2 KO mice. A 300-bp shorter band is observed in DNA from the KO versus WT allele.