Functional characterisation of the CRE/TATA box unit of type 2 deiodinase gene promoter in a human choriocarcinoma cell line.
The regulation of expression of type II deiodinase (D2) is a critical mechanism to maintain appropriate intracellular concentrations of tri-iodothyronine in selected tissues. One of the major regulators of D2 concentrations is cAMP, which potently increases human type II deiodinase (hD2) gene transcription in some tissues via a conserved cAMP response element (CRE) located in the promoter region. In addition, the regulatory region of the hD2 gene contains several TATA box/transcription start site (TSS) units, suggesting the presence of different transcripts that might be characterised by different biological properties. However, it is still unclear whether one ore more TATA box/TSS units are needed in response to cAMP or to other signals able to modulate hD2 transcription. In this study we have analysed the ability of cAMP to regulate hD2 in JEG3 cells, a human choriocarcinoma cell line highly responsive to cAMP. Transient transfection assays of different hD2 gene promoter constructs revealed that cAMP induces transcription starting from the most 5' TSS, located about 80 nucleotides from the CRE. RT-PCR studies have revealed that cAMP activates the expression of a long-lived transcript in JEG3 cells. Site-directed mutagenesis and deletion analysis of promoter constructs have shown that a single CRE/TATA box/TSS unit is needed to confer responsiveness to cAMP. By using chromatin immunoprecipitation studies, we have also demonstrated that the response to cAMP involves the binding of transcription factor CRE binding protein (CREB) to the CRE located in the hD2 promoter. In summary, in JEG3 cells cAMP induces transcription of a long-lived hD2 RNA via CREB and a single CRE/TATA box/TSS unit. This study provides new insights to the regulation of expression of hD2 in placenta.
Laboratory of Molecular and Endocrine Physiopathology, Department of Experimental Medicine and Pathology, University 'La Sapienza' of Rome, Rome, Italy.
Gianluca
G
Canettieri
Antonella
A
Franchi
Rosanna
R
Sibilla
Ernesto
E
Guzmán
Marco
M
Centanni
D001483:Base Sequence; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000201:enzymology* / Q000235:genetics; D000242:Cyclic AMP / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016385:TATA Box; D014158:Transcription, Genetic / Q000502:physiology*; D000097564:Iodothyronine Deiodinase Type II
D000242:Cyclic AMP; D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D007453:Iodide Peroxidase; D000097564:Iodothyronine Deiodinase Type II