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      Unliganded Thyroid Hormone Receptor α Regulates Developmental Timing via Gene Repression in Xenopus tropicalis

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          Abstract

          Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles, and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.

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          Most cited references39

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          Thyroid hormone receptors: multiple forms, multiple possibilities.

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            Efficient TALEN construction and evaluation methods for human cell and animal applications.

            Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.
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              Structural determinants of nuclear receptor assembly on DNA direct repeats.

              Nuclear receptor heterodimers recognize response elements composed of two direct repeats of the consensus sequence 5'-AGGTCA-3' separated by one to five base pairs. The 1.9 A crystal structure of the complex formed by the DNA-binding domains of the 9-cis retinoic acid receptor and thyroid hormone receptor bound to a thyroid-response element shows that the subunits interact through a DNA-supported interface involving the carboxy-terminal extension of the DNA-binding domain of the thyroid hormone receptor. The stereochemistry suggests a mechanism by which heterodimers recognize the inter-half-site spacing between direct repeats.
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                Author and article information

                Journal
                Endocrinology
                Endocrinology
                endo
                endoc
                endo
                Endocrinology
                Endocrine Society (Chevy Chase, MD )
                0013-7227
                1945-7170
                February 2015
                2 December 2014
                2 December 2014
                : 156
                : 2
                : 735-744
                Affiliations
                Department of Biological Sciences (J.C., L.S., D.R.B.), University of Cincinnati, Cincinnati, Ohio 45221; and Department of Mathematical and Life Sciences (K.T.S., T.S., T.Y.), Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan
                Author notes
                Address all correspondence and requests for reprints to: Daniel R. Buchholz, PhD, Department of Biological Sciences, University of Cincinnati, 312 Clifton Court, Cincinnati, OH 45221. E-mail: buchhodr@ 123456ucmail.uc.edu .
                Article
                EN-14-1554
                10.1210/en.2014-1554
                4298327
                25456067
                aa59ad35-36bb-4406-ae11-bc6b0b766e9d

                This article has been published under the terms of the Creative Commons Attribution License (CC-BY: https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright for this article is retained by the author(s). Author(s) grant(s) the Endocrine Society the exclusive right to publish the article and identify itself as the original publisher.

                History
                : 2 July 2014
                : 25 November 2014
                Categories
                Thyroid-TRH-TSH

                Endocrinology & Diabetes
                Endocrinology & Diabetes

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