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Schmitt
Characterization of a thyroid-specific and cyclic adenosine monophosphate-responsive enhancer far upstream from the human sodium iodide symporter gene.Schmitt TL, Espinoza CR, Loos U. Thyroid. 2002 Apr;12(4):273-9. [abstract only]
"We describe the cloning and characterization of a human sodium iodide (NIS) upstream enhancer (NUE). This putative enhancer was cloned based on its sequence homology (69% identity) to the rat NUE. A 296 base pair (bp) genomic DNA fragment, which is located 9000 bp upstream from the human hNIS gene, was amplified by polymerase chain reaction (PCR) and inserted into a luciferase reporter gene in front of both the homologous NIS promoter and the heterologous SV40 promoter. No enhancer activity could be found after transfection into HeLa cells, but in FRTL-5 cells representing the thyroid model, a threefold stimulation of the NIS promoter was found. This enhancer activity was present in both directions and was stimulated threefold by thyrotropin (TSH) and 14-fold by the cyclic adenosine monophosphate (cAMP) agonist forskolin. A small element (TGACGCA) in this enhancer was found to be of central importance, because its site-directed mutagenesis abolished the enhancer activity. This element bound specifically to proteins in nuclear extracts from FRTL-5 cells and to a lesser extent also from HeLa cells. In summary, we describe a thyroid-specific and cAMP-responsive enhancer far upstream from the human NIS gene, which is located in the intronic region of another gene coding for a ribosomal protein."
Transcriptional regulation of the human sodium/iodide symporter gene by Pax8 and TTF-1.Schmitt TL, Espinoza CR, Loos U. Exp Clin Endocrinol Diabetes. 2001;109(1):27-31. [abstract only]
"The regulation of the human Na(+)/I(-) symporter (NIS) gene is of considerable interest for both the diagnosis and therapy of thyroid pathologies. We investigated the influence of the thyroid-specific transcription factors TTF-1 and Pax8 on the NIS promoter and its 5'-flanking sequence. Reporter genes containing the corresponding genomic fragments coupled to a luciferase reporter gene were cotransfected with expression vectors carrying the the cDNA's for TTF-1 and Pax8. Transient transfection assays were performed in HeLa and COS-7 cells, which do not express endogenously these transcription factors. The experiments showed, that TTF-1 had no influence on the NIS promoter. Pax8, on the other hand, had a moderate stimulating effect (threefold) on the proximal NIS promoter. ABCD assays indicated an interaction of in vitro-translated Pax8 with the NIS promoter. However, DNase I footprinting experiments with bacterially expressed Pax8 were negative, suggesting an indirect mode of action with the participation of other proteins. A putative NIS upstream enhancer (NUE) 9000 bp upstream of the NIS gene, which was cloned based on its sequence homology to the rat NUE, was not transactivated by either Pax8 or TTF-1. The present data demonstrate, that the combination of Pax8 and TTF-1 is less important for NIS gene transcription than for other thyroid-specific genes. This is presumably related to the fact, that NIS is expressed also in other tissues such as mammary and salivary gland."
Cloning and characterization of repressory and stimulatory DNA sequences upstream the Na/I-symporter gene promoter.Schmitt TL, Espinoza CR, Loos U. Horm Metab Res. 2000 Jan;32(1):1-5. [abstract only]
"To investigate the existence of potential enhancer or silencer elements in the 5'-flanking region of the human Na+/I-symporter (NIS) gene, we cloned 2,512 bp of genomic DNA further upstream of the previously defined proximal promoter. When tested in reporter gene assays, this sequence had no transcriptional activity per se, but was able to repress the activity of the heterologous SV40 promoter. Conversely, when fused to the homologous NIS gene promoter and thus comprising 3,800 bp 5'-flanking region, the transcription of the proximal NIS promoter was stimulated in the human cell lines FTC-133 (from thyroid) and HeLa, but inhibited in the rat thyroid cell line FRTL-5. This might be due to differences between the upstream regions of the rat and human NIS gene. Comparative analysis with standard promoters (SV40) led to the conclusion that the 5'-flanking region of the human NIS gene also exhibited transcriptional activity in non-thyroid cells. Thyroid-stimulating hormone (TSH) had a moderately stimulating effect on the full length NIS reporter gene construct in FRTL-5 cells. This stimulation is presumably mediated by a putative cAMP responsive element found in the first half of the cloned sequence."
Cloning of a functional promoter of the human sodium/iodide-symporter gene.Behr M, Schmitt TL, Espinoza CR, Loos U. Biochem J. 1998 Apr 15;331 ( Pt 2):359-63.
"We have cloned and sequenced genomic DNA from a human library extending 1300 bp upstream the 5'-untranslated sequence of the cDNA coding for the sodium/iodide symporter. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -443 to -395 relative to the ATG start codon. This minimal promoter, including a putative GC- and TATA- box, was preferentially activated in the rat thyroid cell line FRTL-5, but was also active in non-thyroidal cells, such as COS-7 and Chinese-hamster ovary, albeit to a markedly lower extent."
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