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. 1995 Mar;15(3):1499–1512. doi: 10.1128/mcb.15.3.1499

Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation.

R Uppaluri 1, H C Towle 1
PMCID: PMC230374  PMID: 7862143

Abstract

The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors. The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation. Little is known regarding the structural or functional determinants of these processes. We have utilized genetic screening in the yeast Saccharomyces cerevisiae to identify residues involved in these functions. Random mutations of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated. In this paper we describe the characterization of a class of mutants that exhibit a dissociation between hormone binding and transcriptional activation. These mutants retained hormone binding (> 15% of the wild-type level) yet failed to transactivate a reporter gene. A number of these mutations occurred within the D region, which links the DNA-binding and ligand-binding domains of the receptor. One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process. The remainder retain DNA binding and thus highlight residues critical for receptor activation. In addition, an unexpected group of "superactivator" mutations that led to enhanced hormone-dependent activation in S. cerevisiae were found. These mutations localized to the carboxy-terminal portion of the receptor in a region which contains elements conserved across the superfamily of nuclear receptors. The hormone-dependent phenotype of these superactivator mutations suggests an important role of this segment in ligand-mediated transcriptional activation.

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Selected References

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  1. Ahn B. Y., Livingston D. M. Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol Cell Biol. 1986 Nov;6(11):3685–3693. doi: 10.1128/mcb.6.11.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  3. Baniahmad A., Ha I., Reinberg D., Tsai S., Tsai M. J., O'Malley B. W. Interaction of human thyroid hormone receptor beta with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8832–8836. doi: 10.1073/pnas.90.19.8832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baniahmad A., Steiner C., Köhne A. C., Renkawitz R. Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell. 1990 May 4;61(3):505–514. doi: 10.1016/0092-8674(90)90532-j. [DOI] [PubMed] [Google Scholar]
  5. Baniahmad A., Tsai S. Y., O'Malley B. W., Tsai M. J. Kindred S thyroid hormone receptor is an active and constitutive silencer and a repressor for thyroid hormone and retinoic acid responses. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10633–10637. doi: 10.1073/pnas.89.22.10633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barettino D., Vivanco Ruiz M. M., Stunnenberg H. G. Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBO J. 1994 Jul 1;13(13):3039–3049. doi: 10.1002/j.1460-2075.1994.tb06603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  8. Bodenner D. L., Mroczynski M. A., Weintraub B. D., Radovick S., Wondisford F. E. A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene. J Biol Chem. 1991 Nov 15;266(32):21666–21673. [PubMed] [Google Scholar]
  9. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  10. Brent G. A., Dunn M. K., Harney J. W., Gulick T., Larsen P. R., Moore D. D. Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor. New Biol. 1989 Dec;1(3):329–336. [PubMed] [Google Scholar]
  11. Cama A., Marcus-Samuels B., Taylor S. I. Immunological abnormalities in insulin receptors on cultured EBV-transformed lymphocytes from insulin-resistant patient with leprechaunism. Diabetes. 1988 Jul;37(7):982–988. doi: 10.2337/diab.37.7.982. [DOI] [PubMed] [Google Scholar]
  12. Casanova J., Helmer E., Selmi-Ruby S., Qi J. S., Au-Fliegner M., Desai-Yajnik V., Koudinova N., Yarm F., Raaka B. M., Samuels H. H. Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor. Mol Cell Biol. 1994 Sep;14(9):5756–5765. doi: 10.1128/mcb.14.9.5756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Damm K., Evans R. M. Identification of a domain required for oncogenic activity and transcriptional suppression by v-erbA and thyroid-hormone receptor alpha. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10668–10672. doi: 10.1073/pnas.90.22.10668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Damm K., Thompson C. C., Evans R. M. Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist. Nature. 1989 Jun 22;339(6226):593–597. doi: 10.1038/339593a0. [DOI] [PubMed] [Google Scholar]
  15. Danielian P. S., White R., Lees J. A., Parker M. G. Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J. 1992 Mar;11(3):1025–1033. doi: 10.1002/j.1460-2075.1992.tb05141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fondell J. D., Roy A. L., Roeder R. G. Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression. Genes Dev. 1993 Jul;7(7B):1400–1410. doi: 10.1101/gad.7.7b.1400. [DOI] [PubMed] [Google Scholar]
  18. Garabedian M. J., Yamamoto K. R. Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast. Mol Biol Cell. 1992 Nov;3(11):1245–1257. doi: 10.1091/mbc.3.11.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geffner M. E., Su F., Ross N. S., Hershman J. M., Van Dop C., Menke J. B., Hao E., Stanzak R. K., Eaton T., Samuels H. H. An arginine to histidine mutation in codon 311 of the C-erbA beta gene results in a mutant thyroid hormone receptor that does not mediate a dominant negative phenotype. J Clin Invest. 1993 Feb;91(2):538–546. doi: 10.1172/JCI116233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Giguère V., Hollenberg S. M., Rosenfeld M. G., Evans R. M. Functional domains of the human glucocorticoid receptor. Cell. 1986 Aug 29;46(5):645–652. doi: 10.1016/0092-8674(86)90339-9. [DOI] [PubMed] [Google Scholar]
  21. Glass C. K., Holloway J. M., Devary O. V., Rosenfeld M. G. The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements. Cell. 1988 Jul 29;54(3):313–323. doi: 10.1016/0092-8674(88)90194-8. [DOI] [PubMed] [Google Scholar]
  22. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hall B. L., Smit-McBride Z., Privalsky M. L. Reconstitution of retinoid X receptor function and combinatorial regulation of other nuclear hormone receptors in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6929–6933. doi: 10.1073/pnas.90.15.6929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Himmelfarb H. J., Pearlberg J., Last D. H., Ptashne M. GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators. Cell. 1990 Dec 21;63(6):1299–1309. doi: 10.1016/0092-8674(90)90425-e. [DOI] [PubMed] [Google Scholar]
  25. Ince B. A., Zhuang Y., Wrenn C. K., Shapiro D. J., Katzenellenbogen B. S. Powerful dominant negative mutants of the human estrogen receptor. J Biol Chem. 1993 Jul 5;268(19):14026–14032. [PubMed] [Google Scholar]
  26. Inoue A., Yamakawa J., Yukioka M., Morisawa S. Filter-binding assay procedure for thyroid hormone receptors. Anal Biochem. 1983 Oct 1;134(1):176–183. doi: 10.1016/0003-2697(83)90280-4. [DOI] [PubMed] [Google Scholar]
  27. Jacoby D. B., Zilz N. D., Towle H. C. Sequences within the 5'-flanking region of the S14 gene confer responsiveness to glucose in primary hepatocytes. J Biol Chem. 1989 Oct 25;264(30):17623–17626. [PubMed] [Google Scholar]
  28. Katz R. W., Koenig R. J. Nonbiased identification of DNA sequences that bind thyroid hormone receptor alpha 1 with high affinity. J Biol Chem. 1993 Sep 15;268(26):19392–19397. [PubMed] [Google Scholar]
  29. Krust A., Green S., Argos P., Kumar V., Walter P., Bornert J. M., Chambon P. The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J. 1986 May;5(5):891–897. doi: 10.1002/j.1460-2075.1986.tb04300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kurokawa R., Yu V. C., När A., Kyakumoto S., Han Z., Silverman S., Rosenfeld M. G., Glass C. K. Differential orientations of the DNA-binding domain and carboxy-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev. 1993 Jul;7(7B):1423–1435. doi: 10.1101/gad.7.7b.1423. [DOI] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Lazar M. A. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev. 1993 Apr;14(2):184–193. doi: 10.1210/edrv-14-2-184. [DOI] [PubMed] [Google Scholar]
  33. Lee J. W., Moore D. D., Heyman R. A. A chimeric thyroid hormone receptor constitutively bound to DNA requires retinoid X receptor for hormone-dependent transcriptional activation in yeast. Mol Endocrinol. 1994 Sep;8(9):1245–1252. doi: 10.1210/mend.8.9.7838157. [DOI] [PubMed] [Google Scholar]
  34. Lee M. S., Kliewer S. A., Provencal J., Wright P. E., Evans R. M. Structure of the retinoid X receptor alpha DNA binding domain: a helix required for homodimeric DNA binding. Science. 1993 May 21;260(5111):1117–1121. doi: 10.1126/science.8388124. [DOI] [PubMed] [Google Scholar]
  35. Lee Y., Mahdavi V. The D domain of the thyroid hormone receptor alpha 1 specifies positive and negative transcriptional regulation functions. J Biol Chem. 1993 Jan 25;268(3):2021–2028. [PubMed] [Google Scholar]
  36. Leng X., Tsai S. Y., O'Malley B. W., Tsai M. J. Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor. J Steroid Biochem Mol Biol. 1993 Dec;46(6):643–661. doi: 10.1016/0960-0760(93)90306-h. [DOI] [PubMed] [Google Scholar]
  37. Liu H. C., Towle H. C. Functional synergism between multiple thyroid hormone response elements regulates hepatic expression of the rat S14 gene. Mol Endocrinol. 1994 Aug;8(8):1021–1037. doi: 10.1210/mend.8.8.7997231. [DOI] [PubMed] [Google Scholar]
  38. Mader S., Kumar V., de Verneuil H., Chambon P. Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature. 1989 Mar 16;338(6212):271–274. doi: 10.1038/338271a0. [DOI] [PubMed] [Google Scholar]
  39. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  40. Mixson A. J., Parrilla R., Ransom S. C., Wiggs E. A., McClaskey J. H., Hauser P., Weintraub B. D. Correlations of language abnormalities with localization of mutations in the beta-thyroid hormone receptor in 13 kindreds with generalized resistance to thyroid hormone: identification of four new mutations. J Clin Endocrinol Metab. 1992 Oct;75(4):1039–1045. doi: 10.1210/jcem.75.4.1400869. [DOI] [PubMed] [Google Scholar]
  41. Murray M. B., Zilz N. D., McCreary N. L., MacDonald M. J., Towle H. C. Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors. J Biol Chem. 1988 Sep 5;263(25):12770–12777. [PubMed] [Google Scholar]
  42. Nagaya T., Jameson J. L. Thyroid hormone receptor dimerization is required for dominant negative inhibition by mutations that cause thyroid hormone resistance. J Biol Chem. 1993 Jul 25;268(21):15766–15771. [PubMed] [Google Scholar]
  43. O'Donnell A. L., Koenig R. J. Mutational analysis identifies a new functional domain of the thyroid hormone receptor. Mol Endocrinol. 1990 May;4(5):715–720. doi: 10.1210/mend-4-5-715. [DOI] [PubMed] [Google Scholar]
  44. O'Donnell A. L., Rosen E. D., Darling D. S., Koenig R. J. Thyroid hormone receptor mutations that interfere with transcriptional activation also interfere with receptor interaction with a nuclear protein. Mol Endocrinol. 1991 Jan;5(1):94–99. doi: 10.1210/mend-5-1-94. [DOI] [PubMed] [Google Scholar]
  45. Ohashi H., Yang Y. F., Walfish P. G. Rat liver c-erb A beta 1 thyroid hormone receptor is a constitutive activator in yeast (Saccharomyces cerevisiae): essential role of domains D,E and F in hormone-independent transcription. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1167–1175. doi: 10.1016/0006-291x(91)91015-5. [DOI] [PubMed] [Google Scholar]
  46. Olesen J., Hahn S., Guarente L. Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell. 1987 Dec 24;51(6):953–961. doi: 10.1016/0092-8674(87)90582-4. [DOI] [PubMed] [Google Scholar]
  47. Privalsky M. L., Sharif M., Yamamoto K. R. The viral erbA oncogene protein, a constitutive repressor in animal cells, is a hormone-regulated activator in yeast. Cell. 1990 Dec 21;63(6):1277–1286. doi: 10.1016/0092-8674(90)90423-c. [DOI] [PubMed] [Google Scholar]
  48. Refetoff S. Resistance to thyroid hormone. Clin Lab Med. 1993 Sep;13(3):563–581. [PubMed] [Google Scholar]
  49. Rosen E. D., Beninghof E. G., Koenig R. J. Dimerization interfaces of thyroid hormone, retinoic acid, vitamin D, and retinoid X receptors. J Biol Chem. 1993 Jun 5;268(16):11534–11541. [PubMed] [Google Scholar]
  50. Saatcioglu F., Bartunek P., Deng T., Zenke M., Karin M. A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor). Mol Cell Biol. 1993 Jun;13(6):3675–3685. doi: 10.1128/mcb.13.6.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Saatcioglu F., Deng T., Karin M. A novel cis element mediating ligand-independent activation by c-ErbA: implications for hormonal regulation. Cell. 1993 Dec 17;75(6):1095–1105. doi: 10.1016/0092-8674(93)90319-l. [DOI] [PubMed] [Google Scholar]
  52. Samuels H. H., Stanley F., Casanova J. Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology. 1979 Jul;105(1):80–85. doi: 10.1210/endo-105-1-80. [DOI] [PubMed] [Google Scholar]
  53. Schena M., Freedman L. P., Yamamoto K. R. Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev. 1989 Oct;3(10):1590–1601. doi: 10.1101/gad.3.10.1590. [DOI] [PubMed] [Google Scholar]
  54. Schena M., Picard D., Yamamoto K. R. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 1991;194:389–398. doi: 10.1016/0076-6879(91)94029-c. [DOI] [PubMed] [Google Scholar]
  55. Schräder M., Becker-André M., Carlberg C. Thyroid hormone receptor functions as monomeric ligand-induced transcription factor on octameric half-sites. Consequences also for dimerization. J Biol Chem. 1994 Mar 4;269(9):6444–6449. [PubMed] [Google Scholar]
  56. Schräder M., Müller K. M., Carlberg C. Specificity and flexibility of vitamin D signaling. Modulation of the activation of natural vitamin D response elements by thyroid hormone. J Biol Chem. 1994 Feb 25;269(8):5501–5504. [PubMed] [Google Scholar]
  57. Schräder M., Wyss A., Sturzenbecker L. J., Grippo J. F., LeMotte P., Carlberg C. RXR-dependent and RXR-independent transactivation by retinoic acid receptors. Nucleic Acids Res. 1993 Mar 11;21(5):1231–1237. doi: 10.1093/nar/21.5.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Thompson C. C., Evans R. M. Trans-activation by thyroid hormone receptors: functional parallels with steroid hormone receptors. Proc Natl Acad Sci U S A. 1989 May;86(10):3494–3498. doi: 10.1073/pnas.86.10.3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Toney J. H., Wu L., Summerfield A. E., Sanyal G., Forman B. M., Zhu J., Samuels H. H. Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA. Biochemistry. 1993 Jan 12;32(1):2–6. doi: 10.1021/bi00052a001. [DOI] [PubMed] [Google Scholar]
  60. Umesono K., Evans R. M. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell. 1989 Jun 30;57(7):1139–1146. doi: 10.1016/0092-8674(89)90051-2. [DOI] [PubMed] [Google Scholar]
  61. Vegeto E., Allan G. F., Schrader W. T., Tsai M. J., McDonnell D. P., O'Malley B. W. The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. Cell. 1992 May 15;69(4):703–713. doi: 10.1016/0092-8674(92)90234-4. [DOI] [PubMed] [Google Scholar]
  62. Webster N. J., Green S., Jin J. R., Chambon P. The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell. 1988 Jul 15;54(2):199–207. doi: 10.1016/0092-8674(88)90552-1. [DOI] [PubMed] [Google Scholar]
  63. Weiss R. E., Weinberg M., Refetoff S. Identical mutations in unrelated families with generalized resistance to thyroid hormone occur in cytosine-guanine-rich areas of the thyroid hormone receptor beta gene. Analysis of 15 families. J Clin Invest. 1993 Jun;91(6):2408–2415. doi: 10.1172/JCI116474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wilson T. E., Fahrner T. J., Milbrandt J. The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol. 1993 Sep;13(9):5794–5804. doi: 10.1128/mcb.13.9.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wilson T. E., Paulsen R. E., Padgett K. A., Milbrandt J. Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science. 1992 Apr 3;256(5053):107–110. doi: 10.1126/science.1314418. [DOI] [PubMed] [Google Scholar]
  66. Wrenn C. K., Katzenellenbogen B. S. Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast. J Biol Chem. 1993 Nov 15;268(32):24089–24098. [PubMed] [Google Scholar]
  67. Yen P. M., Sugawara A., Chin W. W. Triiodothyronine (T3) differentially affects T3-receptor/retinoic acid receptor and T3-receptor/retinoid X receptor heterodimer binding to DNA. J Biol Chem. 1992 Nov 15;267(32):23248–23252. [PubMed] [Google Scholar]
  68. Zechel C., Shen X. Q., Chambon P., Gronemeyer H. Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements. EMBO J. 1994 Mar 15;13(6):1414–1424. doi: 10.1002/j.1460-2075.1994.tb06395.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zilz N. D., Murray M. B., Towle H. C. Identification of multiple thyroid hormone response elements located far upstream from the rat S14 promoter. J Biol Chem. 1990 May 15;265(14):8136–8143. [PubMed] [Google Scholar]

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