Characterization of the upstream enhancer of the rat sodium/iodide symporter gene

Summary:

We previously demonstrated the presence of an enhancer that is located between nucleotides - 2264 and - 2495 in the 5′ flanking region of the rat sodium/iodide symporter (NIS) gene ([Ohno et al., 1999]). When attached to NIS or heterologous promoters, this 232 bp fragment, which we call NUE, is able to stimulate transcription in a thyroid-specific and cAMP-dependent manner. A paired-domain transcription factor Pax8 binds to this enhancer and can stimulate the transcription in non-thyroid cells that do not normally support the NUE activities. Cotransfection of PKA, a downstream effector of cAMP, further potentiates the Pax8-mediated transactivation. However, this transcriptional machinery containing pax8 seems to require contributions from the neighboring cis-acting element that is similar to CRE/AP-1 consensus sequences. Modification of this putative CRE/AP-1 site not only represses the NUE transcriptional activities by 90% in FRTL-5 cells, but also nullifies the synergistic effect of PKA on pax8-mediated transactivation in HeLa cells. In this report, we have further characterized the putative CRE/AP-1 site within the NIS upstream enhancer using gel mobility shift assay. An oligonucleotide probe with NIS CRE/AP-1 sequence produced complex binding patterns in both FRTL-5 and HeLa cell, reflecting the presence of diverse classes of binding factors. When compared with CRE or AP-1 elements in other genes, the mobility shift pattern of NIS CRE/AP-1 was similar to those of collagenase TRE, c-Jun TRE, and somatostatin CRE, but the relative intensities of the binding complexes were quite different. This observation raises a possibility that the NIS CRE/AP-site is regulated by a novel mechanism.

Key words:

NIS - AP-1 - cAMP - Rap1 - Pax8

References

• 1 Boussiotis V A, Freeman G J, Berezovskaya A, Barber D L, Nadler L M. Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1.  Science. 278 124-128 1997; 
• 2 Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter.  Nature. 379 458-460 1996; 
• 3 DeCesare D, Vallone D, Caracciolo A, Sassone-Corsi P, Nerlov C, Verde P. Heterodimerization of c-Jun with ATF-2 and c-Fos is required for positive and negative regulation of the human urokinase enhancer.  Oncogene. 11 365-376 1995; 
• 4 de Rooij J, Zwartkruis F J, Verheijen M H, Cool R H, Nijman S M, Wittinghofer A, Bos J L. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.  Nature. 396 474-477 1998; 
• 5 Dremier S, Vandeput F, Zwartkruis F J, Bos J L, Dumont J E, Maenhaut C. Activation of the small G protein Rap1 in dog thyroid cells by both cAMP-dependent and -independent pathways.  Biochem Biophys Res Commun. 267 7-11 2000; 
• 6 Isozaki O, Kohn L D. Control of c-fos and c-myc protooncogene induction in rat thyroid cells in culture.  Mol Endocrinil. 1 839-848 1987; 
• 7 Kawasaki H, Springett G M, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman D E, Graybiel A M. A family of cAMP-binding proteins that directly activate Rap1.  Science. 282 2275-2279 1998; 
• 8 Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T. Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells.  Endocrinology. 138 2227-2232 1997; 
• 9 Kohn L D, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M. The thyrotropin receptor.  Vitam Horm. 50 287-384 1995; 
• 10 Lalli E E, Sassone-Corsi P. Signal transduction and gene regulation: the nuclear response to cAMP.  J Biol Chem. 269 17359-17362 1994; 
• 11 Ohno M, Zannini M, Levy O, Carrasco N, Di Lauro R. The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription.  Mol Cell Biol. 19 2051-2060 1999; 
• 12 Poleev A, Oklandova O, Musti A M, Schneider D, Royer-Pokora B, Plachov D. Determination of functional domains of the human transcription factor pax8 responsible for its nuclear localization and transactivating potential.  Eur J Biochem. 247 860-869 1997; 
• 13 Tazebay U H, Wapnir I L, Levy O, Dohan O, Zuckier L S, Zhao Q H, Deng H F, Amenta P S, Fineberg S, Pestell R G, Carrasco N. The mammary gland iodide transporter is expressed during lactation and in breast cancer.  Nature Med. 6 871-878 2000; 
• 14 Vossler M R, Yao H, York R D, Pan M G, Rim C S, Stork P JS. cAMP activates MAP kinase and Elk-1 through a B-Raf and Rap-1 dependent pathway.  Cell. 89 73-82 1997;