期刊
BIOCHEMICAL JOURNAL
卷 407, 期 -, 页码 101-111出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20070364
关键词
C2BBel cell line; Na+/H+ exchanger (NHE); stimulating protein 1/3 (Sp1/Sp3); transcriptional regulation; 5'-untranslated region
资金
- NIDDK NIH HHS [R01 DK033349, P01-DK067887, R01-DK33349, R01 DK067990, R01-DK67990, P01 DK067887] Funding Source: Medline
We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the -40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, -10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5' flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at - 25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthemore, we identified two positive regulatory elements in the DNA region corresponding to the 5'-UTR (5'-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5'-UTR may co-operate with the 5'-flanking region to achieve the optimal promoter activity.
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