Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element.
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 electrophoretic 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.
Canettieri
G
G
Department of Experimental Medicine and Pathology, University La Sapienza, Rome, Italy.
Celi
F S
FS
Baccheschi
G
G
Salvatori
L
L
Andreoli
M
M
Centanni
M
M
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics*; D008969:Molecular Sequence Data; D009716:Numerical Analysis, Computer-Assisted; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D013724:Teratoma / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000097564:Iodothyronine Deiodinase Type II
D005576:Colforsin; D000242:Cyclic AMP; D017362:Cyclic AMP Response Element-Binding Protein; D007453:Iodide Peroxidase; D000097564:Iodothyronine Deiodinase Type II