We analyzed the structure and function of the 5' flanking region of the human type 2 deiodinase (hD2) gene. Two major transcription start sites were identified at -470/- 474 from the ATG. The 5' flanking region of hD2 gene efficiently directed transcription in transient transfection studies, using luciferase as reporter gene, in HEK 293 cells. Basal transcriptional activity was significantly reduced by deleting the region containing a canonical cAMP-responsive element (CRE) located -766/-759 from ATG. Forskolin treatment significantly increased luciferase activity in cells transfected with CRE-containing constructs. This effect was abolished in constructs that did not contain CRE or contained the mutagenized CRE. Northern blot analysis in JEG-3 cells revealed that the hD2 messenger RNA was markedly increased after stimulation with cAMP agonist. The trophoretic mobility shift assay with hD2-CRE probe and HEK 293 nuclear extract showed the occurrence of a DNA-protein complex, which was competed by specific unlabeled oligonucleotides and supershifted by the anti-CREB and anti-CRE modulator-1 antibodies. A-CREB, a dominant negative inhibitor of CREB, completely inhibited forskolin induction of the hD2 promoter. CREB protein, once cotransfected with hD2 promoter construct: and pKA in F9 teratocarcinoma cells, which are unresponsive to cAMP, was able to stimulate the hD2 gene transcription. These results indicate the existence of a functional promoter within the 5' flanking region of hD2 gene which is characterized by the presence of a CRE. The specific involvement of CREB in the cAMP-mediated hD2 gene promoter induction also has been demonstrated.

Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element / Canettieri, Gianluca; F. S., Celi; G., Baccheschi; L., Salvatori; Andreoli, Mario; Centanni, Marco. - In: ENDOCRINOLOGY. - ISSN 0013-7227. - STAMPA. - 141:5(2000), pp. 1804-1813. [10.1210/en.141.5.1804]

Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element

CANETTIERI, Gianluca;ANDREOLI, Mario;CENTANNI, Marco
2000

Abstract

We analyzed the structure and function of the 5' flanking region of the human type 2 deiodinase (hD2) gene. Two major transcription start sites were identified at -470/- 474 from the ATG. The 5' flanking region of hD2 gene efficiently directed transcription in transient transfection studies, using luciferase as reporter gene, in HEK 293 cells. Basal transcriptional activity was significantly reduced by deleting the region containing a canonical cAMP-responsive element (CRE) located -766/-759 from ATG. Forskolin treatment significantly increased luciferase activity in cells transfected with CRE-containing constructs. This effect was abolished in constructs that did not contain CRE or contained the mutagenized CRE. Northern blot analysis in JEG-3 cells revealed that the hD2 messenger RNA was markedly increased after stimulation with cAMP agonist. The trophoretic mobility shift assay with hD2-CRE probe and HEK 293 nuclear extract showed the occurrence of a DNA-protein complex, which was competed by specific unlabeled oligonucleotides and supershifted by the anti-CREB and anti-CRE modulator-1 antibodies. A-CREB, a dominant negative inhibitor of CREB, completely inhibited forskolin induction of the hD2 promoter. CREB protein, once cotransfected with hD2 promoter construct: and pKA in F9 teratocarcinoma cells, which are unresponsive to cAMP, was able to stimulate the hD2 gene transcription. These results indicate the existence of a functional promoter within the 5' flanking region of hD2 gene which is characterized by the presence of a CRE. The specific involvement of CREB in the cAMP-mediated hD2 gene promoter induction also has been demonstrated.
2000
01 Pubblicazione su rivista::01a Articolo in rivista
Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element / Canettieri, Gianluca; F. S., Celi; G., Baccheschi; L., Salvatori; Andreoli, Mario; Centanni, Marco. - In: ENDOCRINOLOGY. - ISSN 0013-7227. - STAMPA. - 141:5(2000), pp. 1804-1813. [10.1210/en.141.5.1804]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/363413
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